Cross-linked compositions

ABSTRACT

Improved compositions comprising a cross-linkable protein or polypeptide, and a non-toxic material which induces cross-linking of the cross-linkable protein. The compositions are optionally and preferably prepared in a non-phosphate buffer solvent. Optionally and preferably, the cross-linkable protein includes gelatin and any gelatin variant or variant protein as described herein. Optionally and preferably, the non-toxic material comprises transglutaminase (TG), which may optionally comprise any type of calcium dependent or independent transglutaminase, which may for example optionally be a microbial transglutaminase (mTG).

FIELD OF THE INVENTION

The present invention relates to improved cross-linked compositionscomprising a cross-linkable protein and a non-toxic material whichinduces cross-linking of the cross-linkable protein.

BACKGROUND OF THE INVENTION

Biomaterials that can form gels in situ are useful for a variety ofapplications. In many cases, in situ gel-forming materials are used asinjectable matrices for controlled drug delivery or injectable scaffoldsfor tissue engineering. (Gutowska A, Jeong B, Jasionowski M. Anat Rec2001, 263, 342-349. Silva E A, Mooney D J. J Thromb Haemost 2007, 5,590-8. Mahoney M J, Anseth K S. J Biomed Mater Res A 2007, 81, 269-78.)In situ gel-forming materials can also serve as adhesives to bond tissueor seal leaks (either gas or fluid) in a physiological environment.

Interest in soft tissue adhesives is growing because of the desire toreplace or supplement sutures for wound closure (Glickman M, GheissariA, Money S, Martin J, Ballard J. Arch Surg 2002, 137, 326-31; discussion332. Pursifull N F, Morey A F. Curr Opin Urol 2007, 17, 396-401), thetrends toward less invasive and cosmetic surgeries (Tissue Adhesives inClinical Medicine; 2nd ed.; Quinn, J. V., Ed.; B C Decker: Hamilton,Ontario Canada, 2005. Tissue Glue in Cosmetic Surgery; Saltz, R.;Toriumi, D. M., Eds.; Quality Medical Publishing, Inc.,: St. Louis, Mo.,USA 2004), and the need for emergency hemostasis (Pusateri A E, HolcombJ B, Kheirabadi B S, Alam H B, Wade C E, Ryan K L. Journal ofTrauma-Injury Infection and Critical Care 2006, 60, 674-682. Acheson EM, Kheirabadi B S, Deguzman R, Dick E J, Holcomb J B. Journal ofTrauma-Injury Infection and Critical Care 2005, 59, 865-874. KheirabadiB S, Acheson E M, Deguzman R, Sondeen J L, Ryan K L, Delgado A, Dick EJ, Holcomb J B. Journal of Trauma-Injury Infection and Critical Care2005, 59, 25-34.)

In situ gel formation can be initiated by a variety of approaches.Chemical approaches to gel formation include the initiation ofpolymerization either by contact, as in cyanoacrylates, or externalstimuli such as photo-initiation. Also, gel formation can be achieved bychemically crosslinking pre-formed polymers using either low molecularweight crosslinkers such as glutaraldehyde or carbodiimide (Otani Y,Tabata Y, Ikada Y. Ann Thorac Surg 1999, 67, 922-6. Sung H W, Huang D M,Chang W H, Huang R N, Hsu J C. J Biomed Mater Res 1999, 46, 520-30.Otani, Y.; Tabata, Y.; Ikada, Y. Biomaterials 1998, 19, 2167-73. Lim, D.W.; Nettles, D. L.; Setton, L. A.; Chilkoti, A. Biomacromolecules 2008,9, 222-30), or activated substituents on the polymer (Iwata, H.;Matsuda, S.; Mitsuhashi, K.; Itoh, E.; Ikada, Y. Biomaterials 1998, 19,1869-76).

In addition to chemical approaches, gel formation can be achievedthrough physical means using self-assembling peptides (Ellis-Behnke R G,Liang Y X, Tay D K, Kau P W, Schneider G E, Zhang S, Wu W, So K F.Nanomedicine 2006, 2, 207-15. Haines-Butterick L, Rajagopal K, Branco M,Salick D, Rughani R, Pilarz M, Lamm M S, Pochan D J, Schneider J P. ProcNatl Acad Sci USA 2007, 104, 7791-6. Ulijn R V, Smith A M. Chem Soc Rev2008, 37, 664-75).

Finally, biological approaches to initiate gel formation have beeninvestigated based on the crosslinking components from marine adhesives,such as mussel glue (Strausberg R L, Link R P. Trends Biotechnol 1990,8, 53-7), or blood coagulation, as in fibrin sealants (Jackson M R. Am JSurg 2001, 182, 1S-7S. Spotnitz W D. Am J Surg 2001, 182, 8S-14S BuchtaC, Hedrich H C, Macher M, Hocker P, Redl H. Biomaterials 2005, 26,6233-41.27-30).

A variety of biomimetic approaches have also been considered for in situgel formation. In these approaches, polymer crosslinking and gelformation are modeled after one of the crosslinking operations found inbiology. The biological model that has probably attracted the mosttechnological interest is the mussel glue that sets under moistconditions (Silverman H G, Roberto F F. Mar Biotechnol (NY) 2007, 9,661-81. Deacon M P, Davis S S, Waite J H, Harding S E. Biochemistry1998, 37, 14108-12). Cross-linking of the mussel glue is initiated bythe enzymatic conversion of phenolic (i.e., dopa) residues of theadhesive protein into reactive quinone residues that can undergosubsequent inter-protein crosslinking reactions (Burzio L A, Waite J H.Biochemistry 2000, 39, 11147-53. McDowell L M, Burzio L A, Waite J H,Schaefer J J. Biol Chem 1999, 274, 20293-5). A second biologicalcross-linking operation that has served as a technological model is thetransglutaminase-catalyzed reactions that occur during blood coagulation(Ehrbar M, Rizzi S C, Hlushchuk R, Djonov V, Zisch A H, Hubbell J A,Weber F E, Lutolf M P. Biomaterials 2007, 28, 3856-66). Biomimeticapproaches for in situ gel formation have investigated the use of FactorXIIIa or other tissue transglutaminases (Sperinde J, Griffith L.Macromolecules 2000, 33, 5476-5480. Sanborn T J, Messersmith P B, BarronA E. Biomaterials 2002, 23, 2703-10).

One biomimetic approach for in situ gel formation of particular interestis the crosslinking of gelatin by a calcium independent microbialtransglutaminase (mTG). mTG catalyzes an analogous crosslinking reactionas Factor XIIIa but the microbial enzyme requires neither thrombin norcalcium for activity. Initial studies with mTG were targeted toapplications in the food industry (Babin H, Dickinson E. FoodHydrocolloids 2001, 15, 271-276. Motoki M, Seguro K. Trends in FoodScience & Technology 1998, 9, 204-210), while later studies consideredpotential medical applications. Previous in vitro studies have shownthat mTG can crosslink gelatin to form a gel within minutes, thegelatin-mTG adhesive can bond with moist or wet tissue, and the adhesivestrength is comparable to, or better than, fibrin-based sealants (Chen TH, Payne G F, et al. Biomaterials 2003, 24, 2831-2841. McDermott M K,Payne G F, et al. Biomacromolecules 2004, 5, 1270-1279. Chen T, Payne GF, et al. J Biomed Mater Res B Appl Biomater 2006, 77, 416-22).

SUMMARY OF THE INVENTION

The background art does not teach or suggest an improved compositionwhich features one or more additional excipients for controlling one ormore properties of a non-fibrin protein or polypeptide based enzymaticcross-linked material, which could be used for a wide variety ofapplications, such as for hemostatic or body fluid sealing purposes,including but not limited to surgical applications, control ofhemorrhage, sealing of fluid leakage, control of bleeding from a wound.

The present invention provides a composition comprising a cross-linkableprotein or polypeptide and one or more cross-linking materials accordingto at least some embodiments.

According to some embodiments of the present invention, there isprovided a composition comprising a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme which induces cross-linking of said cross-linkableprotein, in a combined acetate and citrate buffer. Optionally, saidcross-linkable protein or polypeptide is present in a sodium acetatebuffer and wherein said enzyme is in a sodium citrate buffer before saidcross-linkable protein or polypeptide and said enzyme are mixed, withtheir respective buffers, to form the composition. Preferably, saidcross-linkable protein or polypeptide comprises gelatin. Morepreferably, said gelatin is at least 250 bloom.

According to some embodiments of the present invention, there isprovided a composition further comprising calcium as anypharmaceutically compatible salt. And optionally further comprisingurea.

Preferably a reduced amount of each of calcium and of urea is presentwhen said calcium and said urea are present in combination than wheneach of calcium or urea is present separately.

More preferably said acetate and/or said citrate is less than about 0.5M.

Optionally and most preferably said acetate and/or said citrate is atleast about 0.01 M.

Optionally an ionic strength is selected from about 0.1 M to about 0.5 Maccording to a desired cross-linking time, wherein an increased ionicstrength leads to a relatively decreased cross-linking time.

Optionally and preferably, for any composition described herein, theenzyme comprises one or more of transglutaminase or a multi-copperoxidase.

More preferably said transglutaminase comprises microbialtransglutaminase.

According to some embodiments of the present invention, there isprovided a composition comprising a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme which induces cross-linking of said cross-linkableprotein, a metal ion and a denaturing agent.

Optionally the cross-linkable protein or polypeptide comprises gelatin.Preferably, said gelatin is at least 250 bloom.

More preferably said metal ion comprises calcium as any pharmaceuticallycompatible salt. Most preferably, said calcium salt comprises one ormore of calcium chloride or calcium hydroxide. Optionally and mostpreferably, said calcium is present in an amount of up to 1M.

Optionally for any composition described herein said denaturing agentcomprises a chaotrope. Preferably, said chaotrope comprises urea. Morepreferably, said urea is present in an amount of up to 4M. Mostpreferably, a reduced amount of each of calcium and of urea is presentwhen said calcium and said urea are present in combination.

Optionally the composition further comprises a calcium sequesteringagent when said metal ion comprises calcium.

According to some embodiments of the present invention, there isprovided a composition comprising a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, a calcium independent enzyme which induces cross-linking of saidcross-linkable protein, and a calcium sequestering agent.

Optionally said calcium sequestering agent comprises one or more ofEDTA, citrate or calgon. Preferably, said citrate or said EDTA ispresent in an amount of from about 0.01 M to about 2M. More preferably,said citrate or said EDTA is present in an amount from about 0.05M toabout 0.2M. Most preferably, said citrate or said EDTA is present in anamount from about 0.5M to about 2M.

According to some embodiments of the present invention, there isprovided a composition comprising a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme which induces cross-linking of said cross-linkableprotein, and a viscosity increasing agent selected from the groupconsisting of Alginate Ester, Gum Arabic, high viscosity Carboxymethylcellulose (CMC), Xanthan Gum, Guar Gum, and PVP.

According to some embodiments of the present invention, there isprovided a composition comprising a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme which induces cross-linking of said cross-linkableprotein, and a kosmotrope.

Optionally said kosmotrope is selected from the group consisting ofproline, trehalose and glutamate or a combination of any two or morethereof. Also optionally the composition further features a chaotrope.

According to some embodiments of the present invention, there isprovided a composition comprising gelatin, transglutaminase and a PEG(polyethylene glycol) derivative capable of covalently binding to saidgelatin. Optionally said PEG derivative comprises any aminated PEGderivative. Preferably, said aminated PEG derivative comprises PEGamine.

According to some embodiments of the present invention, there isprovided a composition comprising gelatin, transglutaminase and a PVA(polyvinyl alcohol) derivative capable of covalently binding to saidgelatin. Optionally, said PVA derivative comprises any aminated PVAderivative. Preferably, said aminated PVA derivative comprises PVAamine.

According to some embodiments of the present invention, there isprovided a composition comprising gelatin, an amine substratecross-linker and an inhibitor of carbamylation. Optionally, saidcross-linker comprises transglutaminase.

Preferably said inhibitor of carbamylation comprises an amine donor.More preferably, said amine donor is selected from the group consistingof glycine and histidine. Optionally and more preferably, said aminedonor is present in an amount that does not inhibit cross-linking ofsaid gelatin by said cross-linker. Also optionally and more preferably,said amine donor is present in an amount to partially inhibitcross-linking of said gelatin by said cross-linker. The abovecompositions may also optionally further comprise urea.

According to some embodiments of the present invention, there isprovided a composition comprising at least partially succinylatedgelatin, non-succinylated gelatin and an amine substrate cross-linker.

According to some embodiments of the present invention, there isprovided a composition comprising at least partially carbamylatedgelatin, non-carbamylated gelatin and an amine substrate cross-linker.

According to some embodiments of the present invention, there isprovided a composition comprising gelatin, a diamine and an aminesubstrate cross-linker. Optionally, said diamine comprises putrescine.

According to some embodiments of the present invention, there isprovided a composition comprising gelatin, an amine donor and an aminesubstrate cross-linker.

Optionally said amine donor comprises polyethylenimine (PEI).Preferably, the amine substrate cross-linker comprises transglutaminase.

According to some embodiments of the present invention, there isprovided a cross-linked composition, comprising a foamed gelatin andtransglutaminase. Optionally, said transglutaminase is present in alyophilized form.

According to some embodiments of the present invention, there isprovided a composition comprising a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, a calcium independent enzyme which induces cross-linking of saidcross-linkable protein, a denaturing agent and an agent for reversing aneffect of said denaturing agent, for reversing sol gel transition pointlowering effect of the denaturing agent.

Optionally said denaturing agent comprises urea and said agent forreversing said effect of said denaturing agent comprises urease.

Optionally any composition as described herein may further comprisesorbitol. Optionally and preferably, said sorbitol is present in asufficient amount to increase the cross-linked composition's flexibilityand/or to accelerate the rate of cross-linking. The composition may alsooptionally further comprise acetate.

According to some embodiments of the present invention, any of thecompositions herein may optionally further comprise a plasticizer.Optionally, said plasticizer is selected from the group consisting ofGum Arabic, Guar Gum, PVA, PEG 6000, Polyvinylpyrrolidone (PVP), citricacid alkyl esters, glycerol esters, phthalic acid alkyl esters, sebacicacid alkyl esters, sucrose esters, sorbitan esters, acetylatedmonoglycerides, glycerols, fatty acid esters, glycols, propylene glycol,lauric acid, sucrose, glyceryl triacetate, poloxamers, diethylphthalate, mono- and di-glycerides of edible fats or oils, dibutylphthalate, dibutyl sebacate, polysorbate, polyethylene glycols 200 to12,000, Carbowax polyethylene glycols, and a surfactant at aconcentration above the CMC (critical micelle concentration) of saidsurfactant; or a combination thereof. Preferably, said surfactantcomprises a polyoxyethylene-sorbitan-fatty acid ester,polyoxyethyleneglycol dodecyl ether, polyoxyethylene-polyoxypropyleneblock copolymer, sodium lauryl sulfate, sodium dodecyl sulfate, sodiumlaureth sulfate, sodium lauryl ether sulfate, poloxamers, poloxamines,alkyl polyglucosides, fatty alcohols, fatty acid salts, cocamidemonoethanolamine, and cocamide diethanolamine.

More preferably, a concentration of said surfactant is in the range offrom about 0.1% to about 5% w/w of dry weight of said cross-linkableprotein. Optionally and most preferably, saidpolyoxyethylene-sorbitan-fatty acid ester comprises one or more ofpolysorbates 20, 21, 0, 60, 61, 65, 80 or 85.

According to some embodiments of the present invention, any of thecompositions herein may optionally further comprise a viscosityincreasing agent selected from the group consisting of Alginate Ester,Gum Arabic, high viscosity Carboxymethyl cellulose (CMC), Xanthan Gum,Guar Gum, and PVP.

According to some embodiments of the present invention, for any of thecompositions herein, optionally said enzyme comprises transglutaminase,the composition further comprising one or more of Cystamine, Cysteine,cyanate or Melanin.

According to some embodiments of the present invention, any of thecompositions herein may optionally further comprise an ammoniascavenging, sequestering or binding agent, a stimulator of ammoniametabolism, or an inhibitor of cellular ammonia uptake. Optionally, saidammonia scavenging agent comprises disaccharide lactulose. Alsooptionally, said ammonia-binding agent comprises a saponin. Preferably,said ammonia scavenger comprises a solution comprising sodiumphenylacetate and sodium benzoate.

Also preferably, said stimulator of ammonia metabolism comprisesL-glutamine, L-glutamate, or a combination thereof.

Also preferably, said inhibitor of cellular ammonia uptake comprisesL-glutamine, L-glutamate, or a combination thereof.

According to some embodiments of the present invention, any of thecompositions herein may optionally further comprise a buffer selectedfrom the group consisting of succinate buffer, maleate buffer,tris(hydroxymethyl)methylamine (TRIS),3-{[(tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),N,N-bis(2-hydroxyethyl)glycine (bicine),N-tris(hydroxymethyl)methylglycine (tricine),2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinicacid, N-(2-hydroxyethyl)piperazine-N′-(2-ethane sulfonic acid) (HEPES),and 2-(N-morpholino)ethanesulfonic acid (MES).

According to some embodiments of the present invention, any of thecompositions herein may optionally have a pH in a range of from about 6to about 7, which is also optionally about 6.

According to some embodiments of the present invention, for any of thecompositions herein may optionally, said enzyme comprisestransglutaminase. Optionally, said transglutaminase is calciumindependent. Preferably, said transglutaminase is microbialtransglutaminase. More preferably, a protein concentration of saidtransglutaminase is present in an amount from about 0.0001% to about 2%w/w of the composition. Most preferably, said transglutaminase ispresent in an amount of from about 0.01% to about 1.35% w/w of thecomposition. Also most preferably, said transglutaminase is present inan amount of from about 0.05% to about 0.5% w/w of the composition.

Optionally and most preferably said transglutaminase is present in anamount of from about 0.1% to about 0.4% w/w of the composition.

Optionally said concentration of transglutaminase is in the range offrom about 1 to about 180 enzyme units (U/mL) of total composition.Preferably said concentration of transglutaminase is in the range offrom about 4 to about 70 enzyme units (U/mL) of total composition. Morepreferably, said concentration of transglutaminase is in the range offrom about 10 to about 55 enzyme units (U/mL) of total composition.

Optionally for any of the compositions herein a ratio of cross linkingmaterial:cross linkable protein solution is about 1:1 to 1:5 v/v.

According to some embodiments of the present invention, for any of thecompositions herein, said cross-linkable protein or polypeptidecomprises gelatin and wherein said gelatin is produced from animalorigin, recombinant origin or a combination thereof. Optionally saidanimal origin is selected from the group consisting of fish and mammals.Preferably, said mammal is selected from the group consisting of pigsand cows. More preferably, said gelatin is of type A (Acid Treated) orof type B (Alkaline Treated). Most preferably, said gelatin compriseshigh molecular weight gelatin. Optionally and most preferably, saidgelatin is at least about 250 bloom. Also optionally and mostpreferably, said gelatin is produced during the first extraction.

According to some embodiments of the present invention, any of thecompositions herein may optionally further comprise a method formanufacturing a composition for cross-linking, comprising: preparing asolution of a cross-linkable protein or polypeptide, with the provisothat said protein or polypeptide is not fibrin, an enzyme forcross-linking said cross-linkable protein or polypeptide, and apharmaceutically acceptable calcium salt; and adding a calciumsequestering agent to said solution.

Optionally said calcium sequestering agent comprises one or more of apolyphosphate salt, and a carboxylate, or combinations thereof. Alsooptionally, said polyphosphate salt is selected from the groupconsisting of a pyrophosphate, a tripolyphosphate, a higherpolyphosphate salt, and a hexametaphosphate salt, or combinationsthereof. Preferably, the pyrophosphate is selected from the groupconsisting of tetrasodium pyrophosphate, disodium dihydrogenpyrophosphate, tetrapotassium pyrophosphate, dipotassium dihydrogenpyrophosphate, and dipotassium disodium pyrophosphate, or combinationsthereof. Also preferably, said tripolyphosphate is selected from thegroup consisting of pentasodium tripolyphosphate, and pentapotassiumtripolyphosphate, or combinations thereof.

Also preferably, said carboxylate is selected from the group consistingof an alkali metal citrate salt, an alkali metal acetate salt, an alkalimetal lactate salt, an alkali metal tartrate salt, an alkali metalmalate salt, an alkali metal salt of ethylenediaminetetraacetic acid,and editronic acid, or combinations thereof. More preferably, saidcarboxylate is selected from the group consisting ofethylenediaminetetraacetic acid and sodium citrate, or a combinationthereof.

Optionally and more preferably, said hexametaphosphate comprises sodiumhexametaphosphate.

According to some embodiments of the present invention, there isprovided a method for manufacturing a composition for cross-linking,comprising: preparing a solution of a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme for cross-linking said cross-linkable protein orpolypeptide, and a denaturant; and adding an agent for reversing aneffect of said denaturing agent, for reversing sol gel transition pointlowering effect of the denaturing agent, to said solution.

Optionally said denaturing agent comprises urea and said agent forreversing said effect of said denaturing agent comprises urease.

According to some embodiments of the present invention, there isprovided a method for manufacturing a composition for cross-linking,comprising: preparing a solution of a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme for cross-linking said cross-linkable protein orpolypeptide, and a chaotrope; and adding a kosmotrope to said solution.

Optionally, wherein said kosmotrope is selected from the groupconsisting of proline, trehalose and glutamate or a combination of anytwo or more thereof.

According to some embodiments of the present invention, there isprovided a method for manufacturing a composition for cross-linking,comprising: preparing a solution of a cross-linkable protein orpolypeptide, with the proviso that said protein or polypeptide is notfibrin, and transglutaminase for cross-linking said cross-linkableprotein or polypeptide; and adding one or more of Cystamine, Cysteine,cyanate or Melanin to said solution for at least partial inhibition ofsaid transglutaminase.

According to some embodiments of the present invention, there isprovided a microbial transglutaminase composition with specificactivity >25 enzyme units per milligram, >95% electrophoretic purity, <5endotoxin units per gram, and <10 CFU/g. Such a transglutaminase mayoptionally be provided as the cross-linker of any of the above claims.

According to some embodiments of the present invention, the compositionfeatures a buffer and one or more other excipients, which are selectedto overcome at least some of the drawbacks of the background art.

Optionally and preferably, the buffer is a non-phosphate buffer, whichis optionally and more preferably selected from the group consisting ofan acetate buffer (such as sodium acetate), citrate buffer (such assodium citrate), succinate buffer, maleate buffer,tris(hydroxymethyl)methylamine (TRIS),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),N,N-bis(2-hydroxyethyl)glycine (bicine),N-tris(hydroxymethyl)methylglycine (tricine),2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinicacid, N-(2-hydroxyethyl)piperazine-N′-(2-ethane sulfonic acid) (HEPES),and 2-(N-morpholino)ethanesulfonic acid (MES).

According to some embodiments, the composition is used as a vehicle forlocalized drug delivery.

According to some embodiments, the composition is an injectable scaffoldfor tissue engineering.

According to some embodiments, the composition is a hemostaticcomposition. According to some embodiments, the composition is a bodyfluid sealing composition.

The compositions of the present invention preferably provide rapidhemostasis, thereby minimizing blood loss following injury or surgery.

“Wound” as used herein refers to any damage to any tissue of a patientthat results in the loss of blood from the circulatory system or theloss of any other bodily fluid from its physiological pathway, such asany type of vessel. The tissue can be an internal tissue, such as anorgan or blood vessel, or an external tissue, such as the skin. The lossof blood or bodily fluid can be internal, such as from a ruptured organ,or external, such as from a laceration. A wound can be in a soft tissue,such as an organ, or in hard tissue, such as bone. The damage may havebeen caused by any agent or source, including traumatic injury,infection or surgical intervention. The damage can be life-threateningor non-life-threatening.

“TG” refers to transglutaminase of any type; “mTG” may also refer tomicrobial transglutaminase and/or to any type of transglutaminase,depending upon the context (in the specific experimental Examples below,the term refers to microbial transglutaminase). Optionally, thetransglutaminase comprises a plant, recombinant animal, or microbederived transglutaminase other than blood derived Factor XIII; however,the actual production process for the transglutaminase may optionallycomprise any type of recombinant method as is known in the art.

Surgical wound closure is currently achieved by sutures and staples thatfacilitate healing by pulling tissues together. However, very often theyfail to produce the adequate seal necessary to prevent fluid leakage.Thus, there is a large, unmet medical need for devices and methods toprevent leakage following surgery, including leaks that frequently occuralong staple and suture lines. Such devices and methods are needed as anadjunct to sutures or staples to achieve hemostasis or otherfluid-stasis in peripheral vascular reconstructions, durareconstructions, thoracic, cardiovascular, lung, neurological, andgastrointestinal surgeries. Most high-pressure hemostatic devicescurrently on the market are nominally, if at all adhesive. Good examplesof such devices are the QuikClot® ACS™ (Z-Medica, Wallington, Conn.) andHemCon™ bandage (HemCon, Portland, Oreg.), the two hemostatic devicescurrently supplied to members of the US armed forces. The chitosannetwork that makes up the HemCon bandage can be saturated with blood andfail quickly when faced with brisk flood flow or after 1-2 hours whenconfronted with moderate blood flow from a wound (B. S Kheirabadi et al.(2005). J. Trauma. 59:25-35; A. E. Pusateri et al. (2006). J. Trauma.60:674-682). The QuikClot minerals must cause a dangerous amount of heatin order to be effective (A. E. Pusateri et al. (2006). J. Trauma.60:674-682).

Other polysaccharide-based hemostatic devices that have been suggestedfor use in hemorrhage control are RDH™ (Acetyl Glucosamine), TraumaDEX™(MPH), and Chitoskin™ (Chitosan & Gelatin). However, none of these typesof bandages have been able to consistently demonstrate avoidance offailure in the face of significant blood flow. Other recently introducedhemostatic devices include Celox™ (Chitosan Crystals) and WoundStat™(TraumaCure Inc., MD) (granular blend of smectite mineral and a superabsorbent polymer). However, both of these products rapidly swell tofill wound sites, making them appropriate only for accelerating bloodclotting in specific types of wounds and presenting a danger of reducingor even eliminating blood flow in surrounding blood vessels.

All of the above-mentioned products rely on the natural clotting cascadeto control fluid leakage from a wound site. As such, they aresignificantly limited in their capacity. General wound site sealing,particularly of injured sites leaking non-blood fluids, is beyond thescope of these products.

With regard to previous efforts to form a tissue adhesive from thecrosslinking of gelatin with mTG (ie McDermott et al 2004 and Chen etal. 2006), the commercial application of these efforts have been limiteddue to the thermoreversible gelation that takes place in gelatinsolutions at operating room temperatures. Though urea has been proposedin the past for lowering the gelation transition point of gelatinsolutions for use in adhesives (Otani et al. 1998), this was notpreviously considered for use with gelatin-mTG sealants since urea hasbeen established as a strong denaturant that can significantly disruptenzyme activity (Rajagopalan et al. J Biologica Chem 236(4), 1961).Furthermore, in studies that specifically explored mTG activity in thepresence of urea, it was found that very low concentrations of urea(<0.5M) severely inhibit mTG function (Nomura Y et al. Biosci BiotechBiochem 65(4), 2001: p. 982-985) and high concentrations of urea (8M)completely inactivate mTG (Yokoyama K et al. Protein Exp & Purif 26,2002: p. 329-335 2002).

Surprisingly, the present inventors found that urea could in fact besuccessfully used with gelatin and mTG under certain circumstances, asdescribed in PCT Pub. No. WO/2008/076407, filed on Dec. 17, 2007, by thepresent inventors, hereby incorporated by reference as if fully setforth herein. Further surprisingly, the present inventors have expandedthe potential use and inclusion of urea in a crosslinked gelatin/mTGcomposition as described in greater detail below.

As used herein, “about” means plus or minus approximately ten percent ofthe indicated value.

Other features and advantages of the various embodiments of theinvention will be apparent from the following detailed description, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a graph showing pH stability of mTG;

FIG. 2 is a graph showing viscosity changes with time of mTG in 0.5MSodium Acetate (Na—Ac) and 0.5M Sodium Citrate;

FIG. 3A-F shows the time to viscosities of 3×106 cP (30% of fully formedgel) and 9×106 cP (90% of fully formed gel) for different illustrativeformulations according to some embodiments of the present invention;

FIG. 4 shows the results of gel electrophoresis of the purified mTGmaterial (lanes 2-8);

FIG. 5 shows the relative cross linking rate of different plasticizersolutions compared to control;

FIG. 6 shows results of some illustrative gelatin solutions according tosome embodiments of the present invention: control, A and B;

FIGS. 7A, 7B and 7C summarize results of various illustrative gelatinsolutions according to some embodiments of the present invention withvarious Glycine and Histidine additives, functioning as Carbamylationinhibitors, which were crosslinked and tested by viscometer afterdifferent incubation times;

FIG. 8 summarizes results of control solutions with and without Cyanateadditives according to some embodiments of the present invention whichwere crosslinked and tested by viscometer after different incubationtimes of high temperature; and

FIG. 9 shows schematics of the burst pressure system and the associatedtissue manifold.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of improved compositions comprising a solutionof a cross-linkable protein or polypeptide, and a solution of one ormore non-toxic materials which induces cross-linking of thecross-linkable protein.

According to some embodiments, the solution of a cross-linkable proteinor polypeptide, and a solution of one or more non-toxic materials whichinduces cross-linking of the cross-linkable protein are prepared in anon-phosphate buffer solvent.

Optionally and preferably, the cross-linkable protein includes gelatinand any gelatin variant or variant protein as described herein.Optionally and preferably, the non-toxic material comprisestransglutaminase (TG), which may optionally comprise any type of calciumdependent or independent transglutaminase, which may for exampleoptionally be a calcium-independent microbial transglutaminase (mTG).Without wishing to be limited in any way, among the improved propertiesof at least some embodiments of the present invention, the compositionsof the present invention provide an increased rate of proteincross-linking as compared to background art compositions. Furthermore,the crosslinking reaction of mTG represents a significant improvementover that catalyzed by Factor XIIIa of the blood coagulation system.Unlike Factor XIIIa, the microbial enzyme requires neither thrombin norcalcium for activity.

The present invention therefore, in at least some embodiments, providesan improved application of a gelatin-mTG adhesive composition for avariety of soft tissue applications. The present invention furtherprovides, in some embodiments, a method for preparing a composition, themethod comprising providing a solution of a cross-linkable protein orpolypeptide; providing a solution of one or more cross-linkingmaterials; and mixing the solution of the cross-linkable protein orpolypeptide with the solution of cross-linking materials.

According to some embodiments of the present invention, the compositionis provided in a bandage, which is preferably adapted for use as ahemostatic bandage. The herein described compositions may additionallyhave one or uses including but not limited to tissue adhesives(particularly biomimetic tissue adhesives), tissue culture scaffolds,tissue sealants, hemostatic compositions, drug delivery platforms,surgical aids, or the like, as well as other non-medical uses, includingbut not limited to edible products, cosmetics and the like, such as, forexample, in purification of enzymes for use in food products.

Various embodiments of the present invention are described in greaterdetail below, under section headings which are provided for the sake ofclarity only and without any intention of being limiting in any way.

Cross-Linkable Protein

According to a preferred embodiment of the present invention, thecross-linkable protein comprises gelatin.

Gelatin may optionally comprise any type of gelatin which comprisesprotein that is known in the art, preferably including but not limitedto gelatin obtained by partial hydrolysis of animal tissue and/orcollagen obtained from animal tissue, including but not limited toanimal skin, connective tissue (including but not limited to ligaments,cartilage and the like), antlers or horns and the like, and/or bones,and/or fish scales and/or bones or other components; and/or arecombinant gelatin produced using bacterial, yeast, animal, insect, orplant systems or any type of cell culture.

According to preferred embodiments of the present invention, gelatinfrom animal origins preferably comprises gelatin from mammalian originsand more preferably comprises one or more of pork skins, pork and cattlebones, or split cattle hides, or any other pig or bovine source. Morepreferably, such gelatin comprises porcine gelatin since it has a lowerrate of anaphylaxis. Gelatin from animal origins may optionally be oftype A (Acid Treated) or of type B (Alkaline Treated), though it ispreferably type A (however an example using type B gelatin is givenbelow).

Preferably, gelatin from animal origins comprises gelatin obtainedduring the first extraction, which is generally performed at lowertemperatures (50-60° C., although this exact temperature range is notnecessarily a limitation). Gelatin produced in this manner will be inthe range of 250-300 bloom and has a high molecular weight of at leastabout 95-100 kDa. Preferably, 275-300 bloom gelatin is used.

A non-limiting example of a producer of such gelatins is PB Gelatins(Tessenderlo Group, Belgium).

According to some embodiments of the present invention, gelatin fromanimal origins optionally comprises gelatin from fish. Optionally anytype of fish may be used, preferably a cold water variety of fish suchas carp, cod, or pike, or tuna. The pH of this gelatin (measured in a10% solution) preferably ranges from 4-6.

Cold water fish gelatin forms a solution in water at 10° C. and thus allcold water fish gelatin are considered to be 0 bloom. For the presentinvention, a high molecular weight cold water fish gelatin is optionallyand preferably used, more preferably including a molecular weight of atleast about 95-100 kDa. This is equivalent to the molecular weight of a250-300 bloom animal gelatin. Cold water fish gelatin undergoesthermoreversible gelation at much lower temperatures than animal gelatinas a result of its lower levels of proline and hydroxyproline. Per 1000amino acid residues, cold water fish gelatin has 100-130 proline and50-75 hydroxyproline groups as compared to 135-145 proline and 90-100hydroxyproline in animal gelatins (Haug I J, Draget K I, Smidsrød O.(2004). Food Hydrocolloids. 18:203-213).

A non-limiting example of a producer of such a gelatin is NorlandProducts (Cranbury, N.J.).

In some embodiments of the present invention, low endotoxicity gelatinis used to form the gelatin solution component of the gelatin-mTGcomposition. Such a gelatin is available commercially from supplierssuch as Gelita™ (Eberbach, Germany). Low endotoxicity gelatin is definedas gelatin with less than 1000 endotoxicity units (EU) per gram. Morepreferably, gelatin of endotoxicity less than 500 EU/gram is used.

For very high sensitivity applications, such as with materials that willcome into contact with either the spine or the brain, gelatin withendotoxicity of less than 100 EU/gram is preferred, gelatin with lessthan 50 EU/g is more preferred. Gelatin with endotoxicity less than 10EU/g is very expensive but could also be used as part of at least someembodiments of the present invention in sensitive applications.

According to some embodiments of the present invention, type I, type II,or any other type of hydrolyzed or non-hydrolyzed collagen replacesgelatin as the protein matter being cross-linked. Various types ofcollagen have demonstrated the ability to form thermally stablemTG-crosslinked gels (O'Halloran D M, et al. Characterization of amicrobial transglutaminase cross-linked type II collagen scaffold.Tissue Eng. 2006 June; 12(6):1467-74. Garcia Y, et al. Assessment ofcell viability in a three-dimensional enzymatically cross-linkedcollagen scaffold. J Mater Sci Mater Med. 2007 October;18(10):1991-2001. Epub 2007 Jun. 7. Nomura Y, et al. Improvement ofshark type I collagen with microbial transglutaminase in urea. BiosciBiotechnol Biochem. 2001 April; 65(4):982-5.)

According to some embodiments of the present invention, a recombinanthuman gelatin is used. Such a gelatin is available commercially fromsuppliers such as Fibrogen™ (San Francisco, Calif.). Recombinant gelatinis preferably at least about 90% pure and is more preferably at leastabout 95% pure. Some recombinant gelatins are non-gelling at 10° C. andthus are considered to be 0 bloom. For some embodiments of the presentinvention, a high molecular weight recombinant gelatin is preferablyused, more preferably including a molecular weight of at least about95-100 kDa.

As noted above, the cross-linkable protein preferably comprises gelatinbut may also, additionally or alternatively, comprise another type ofprotein. According to some embodiments of the present invention, theprotein is also a substrate for transglutaminase, and preferablyfeatures appropriate transglutaminase-specific polypeptide and polymersequences. These proteins may optionally include but are not limited tosynthesized polymer sequences that independently have the properties toform a bioadhesive or polymers that have been more preferably modifiedwith transglutaminase-specific substrates that enhance the ability ofthe material to be cross-linked by transglutaminase. Non-limitingexamples of each of these types of materials are described below.

Synthesized polypeptide and polymer sequences with an appropriatetransglutaminase target for cross-linking have been developed that havetransition points preferably from about 20 to about 40° C. Preferredphysical characteristics include but are not limited to the ability tobind tissue and the ability to form fibers. Like gelling type gelatins(described above), these polypeptides may optionally be used incompositions that also feature one or more substances that lower theirtransition point.

Non-limiting examples of such peptides are described in U.S. Pat. Nos.5,428,014 and 5,939,385, both filed by ZymoGenetics Inc, both of whichare hereby incorporated by reference as if fully set forth herein. Bothpatents describe biocompatible, bioadhesive, transglutaminasecross-linkable polypeptides wherein transglutaminase is known tocatalyze an acyl-transfer reaction between the γ-carboxamide group ofprotein-bound glutaminyl residues and the ε-amino group of Lys residues,resulting in the formation of ε-(γ-glutamyl) lysine isopeptide bonds.

For example, polypeptides having 13-120 amino acid residues aredescribed, comprising a segment of the formula S1-Y-S2, wherein: S1 isThr-Ile-Gly-Glu-Gly-Gln; Y is a spacer peptide of 1-7 amino acids or notpresent; and S2 is Xaa-Lys-Xaa-Ala-Gly-Asp-Val. Optionally, the spacerpeptide Y is Gln-His-His-Leu-Gly, Gln-His-His-Leu-Gly-Gly orHis-His-Leu-Gly-Gly. Also optionally, Xaa, amino acid 1, of S2 is Ala orSer. Optionally, the spacer peptide comprises His-His-Leu-Gly.Optionally and preferably, at least one of Y and S2 are free of Glnresidues. Optionally, the carboxyl terminal amino acid residue of thepolypeptide is Pro or Gly. Specific non-limiting examples of thepolypeptides include the following:Thr-Ile-Gly-Glu-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val,Thr-Ile-Gly-Glu-Gly-Gln-Gln-His-His-Leu-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val,Thr-Ile-Gly-Glu-Gly-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val,or Leu-Ser-Gln-Ser-Lys-Val-Gly. The patent also describes high molecularweight, biocompatible, bioadhesive, transglutaminase-cross-linkablecopolymers and homopolymers involving these peptides.

U.S. Pat. No. 5,939,385 describes biocompatible, bioadhesivetransglutaminase cross-linkable polypeptides. These polypeptidespreferably have about 9-120 amino acid residues comprising a segment ofthe formula S1-Y-S2, wherein: S1 is selected from the group consistingof Ile-Gly-Glu-Gly-Gln, Gly-Glu-Gly-Gln, Glu-Gly-Gln, and Gly-Gln; Y isHis-His-Leu-Gly-Gly or His-His-Leu-Gly; and S2 is selected from thegroup consisting of Ala-Lys-Gln-Ala-Gly-Asp, Ala-Lys-Gln-Ala-Gly,Ala-Lys-Gln-Ala, Ala-Lys-Gln, Ala-Lys-Ala-Gly-Asp-Val, Ala-Lys-Ala andAla-Lys, wherein said polypeptide has an amino-terminus and acarboxy-terminus and is cross-linkable by a transglutaminase. Apreferred polypeptide is Gly-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln. Alsopreferred is a polypeptide wherein the polypeptide is flanked on eitheror both the amino-terminus and the carboxy-terminus by an elastomericpolypeptide. It further provides an elastomeric polypeptide wherein theelastomeric polypeptide is a pentapeptide or a tetrapeptide,particularly a flanked polypeptide wherein the flanking elastomericpolypeptide is Val-Pro-Gly-Val-Gly, Ala-Pro-Gly-Val-Gly,Gly-Val-Gly-Val-Pro, Val-Pro-Gly-Gly or any portion thereof, preferablysuch that the amino-terminus of the flanked polypeptide is Val and thecarboxy-terminus of the flanked polypeptide is Gly. The patent alsodescribes high molecular weight, biocompatible, bioadhesive,transglutaminase-cross-linkable copolymers and homopolymers involvingthese peptides.

Cross-Linking Material

Optionally and preferably, the non-toxic cross-linking materialcomprises transglutaminase (TG), which may optionally comprise any typeof calcium dependent or independent transglutaminase (mTG), which mayfor example optionally be a microbial transglutaminase.

According to some embodiments of the present invention, newly availablecommercial transglutaminase products containing 10% or more mTG may beused. Non-limiting examples of commercially available transglutaminaseproducts of this sort include those produced by Ajinomoto Co. (Kawasaki,Japan) and Yiming Chemicals (China). A non-limiting example of such aproduct from this company is Activa TG—Ingredients: mTG (10%) andmaltodextrin (90%); Activity: 810-1,350 U/g of Activa. Non-limitingexamples of such products from Yiming include one product containing 10%mTG and 90% maltodextran and one product containing 10% mTG and 90%lactose, also of activity 810-1,350 U/g of product. Other non-limitingexamples of such products from Yiming include one product containing 30%mTG and 70% maltodextran and one product containing 30% mTG and 70%lactose, both with activity 2,430-4,050 U/g of product.

As noted above, the cross-linking material preferably comprisestransglutaminase but may also, additionally, comprise another type ofcross-linking material according to some embodiments of the presentinvention.

Non-limiting examples of such cross-linking agents include carbodiimidessuch as N,N-(3-(dimethylamino)propyl)-N-ethyl carbodiimide (EDC),N-hydroxysuccinimide (NHS) with EDC, or carbodiimides used together withpoly(L-glutamic acid) (PLGA) and polyacrylic acid. In anotherembodiment, such cross-linking agents can include Tyrosinase orTyrosinase with chitosan. In another embodiment, cross-linking(polymerization) is photo-initiated with ultraviolet light or γ-rays. Inanother embodiment, cross-linking agents can include alkylene, citricacid (carbonic acid), or Nano-hydroxyapatite (n-HA)+poly(vinyl alcohol)(PVA).

In another embodiment, a cross-linking agent is a plant-derivedpolyphenol such as (i.e. hydroxylated cinnamic acids, such as caffeicacid (3,4-dihydroxycinnamic acid), chlorogenic acid (preferably thequinic acid ester), caftaric acid (preferably the tartaric acid ester),and flavonoids (i.e. as quercetin and rutin). In another embodiment, theadditional cross-linking agent is an oxidized mono or disaccharide,oxo-lactose, or a dialdehyde based on a sugar moiety(galacto-hexodialdose) (GALA). In another embodiment, Genipin or otheriridoid glycoside derivative, or Secoiridoids, preferable oleuropein,comprises the cross-linking agent. In another embodiment, thecross-linking agent is a thiol-reactive poly(ethylene glycol). Inanother embodiment, the cross-linking agent is dextran, oxidizeddextran, dextran dialdehyde. In another embodiment, the cross-linkingagent is a multi-copper oxidase such as laccase or bilirubin oxidase.

Illustrative Compositions

The above described cross-linking substrates and cross-linking materialsmay optionally be combined with one or more additional materials to formvarious compositions according to the present invention. According tosome embodiments, the adhesive material optionally and preferablycomprises: (i) gelatin; (ii) a transglutaminase. More preferably, thegelatin and transglutaminase are provided in sufficient quantities to beuseful as a sealing, hemostatic agent.

In addition, one or more supplements can also be contained in thehemostatic product, e.g., drugs such as growth factors, polyclonal andmonoclonal antibodies and other compounds. Illustrative examples of suchsupplements include, but are not limited to: antibiotics, such astetracycline and ciprofloxacin, amoxicillin, and metronidazole;anticoagulants, such as activated protein C, heparin, prostracyclin(PGI₂), prostaglandins, leukotrienes, antitransglutaminase III, ADPase,and plasminogen activator; steroids, such as dexamethasone, inhibitorsof prostacyclin, prostaglandins, leukotrienes and/or kinins to inhibitinflammation; cardiovascular drugs, such as calcium channel blockers,vasodilators and vasoconstrictors; chemoattractants; local anestheticssuch as bupivacaine; and antiproliferative/antitumor drugs such as5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such asgangcyclovir, zidovudine, amantidine, vidarabine, ribaravin,trifluridine, acyclovir, dideoxyuridine and antibodies to viralcomponents or gene products; cytokines, such as alpha- or beta- orgamma-Interferon, alpha- or beta-tumor necrosis factor, andinterleukins; colony stimulating factors; erythropoietin; antifungals,such as diflucan, ketaconizole and nystatin; antiparasitic agents, suchas pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsinand alpha-1-antichymotrypsin; anesthetics, such as bupivacaine;analgesics; antiseptics; and hormones. Other illustrative supplementsinclude, but are not limited to: vitamins and other nutritionalsupplements; glycoproteins; fibronectin; peptides and proteins;carbohydrates (both simple and/or complex); proteoglycans;antiangiogenins; antigens; lipids or liposomes; and oligonucleotides(sense and/or antisense DNA and/or RNA).

Buffer Selection

PCT Pub. No. WO/2008/076407, filed on Dec. 17, 2007, disclosescompositions comprising gelatin as a cross-linkable protein, andmicrobial transglutaminase as a non-toxic cross-linking material,wherein Phosphate Buffered Saline (PBS) is used as a preferred solventfor dissolving both the gelatin and the mTG. However, PBS has beendiscovered to reduce the speed of mTG-facilitated gelatin cross-linking.Without wishing to be limited to a single hypothesis, it is suggestedthat the phosphate molecules in the buffer reduce the cross-linkingactivity of the mTG enzyme. Therefore, buffers which are devoid ofphosphate have been determined to be more efficacious for use as buffersof both gelatin and mTG solutions.

Non-limiting examples of non-phosphate buffers suitable for use in thepresent invention include acetate buffer (such as sodium acetate),citrate buffer (such as sodium citrate), succinate buffer, maleatebuffer, tris(hydroxymethyl)methylamine (TRIS),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),N,N-bis(2-hydroxyethyl)glycine (bicine),N-tris(hydroxymethyl)methylglycine (tricine),2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinicacid, N-(2-hydroxyethyl)piperazine-N′-(2-ethane sulfonic acid) (HEPES),and 2-(N-morpholino)ethanesulfonic acid (MES).

Optionally and preferably, buffers comprising one or more carboxylgroups are used. More preferably, the buffer comprises sodium acetate orsodium citrate. More preferably, sodium acetate is used as a buffer forthe protein or polypeptide, and sodium citrate or sodium acetate is usedas buffer for the cross-linking material.

Optionally and preferably, the buffer solutions have a concentration inthe range of from about 0.01 to about 0.6M. More preferably, the buffersolution for the protein or polypeptide has a concentration in the rangeof from about 0.05 to about 0.15M, and most preferably has aconcentration of about 0.1M. More preferably, the buffer solution forthe cross-linking material has a concentration in the range of fromabout 0.1 to about 0.5M. The benefit of these concentrations isdescribed with regard to the below illustrative, non-limiting examples.

Without wishing to be limited by a single hypothesis, sodium acetatebuffer is believed to accelerate the mTG-catalyzed crosslinking of theprotein, as described with regard to the below illustrative,non-limiting examples.

Without wishing to be limited by a single hypothesis, sodium citratebuffer is believed to improve the mechanical and biocompatibilityproperties of the crosslinked composition if added to theprotein/polypeptide solution together with the crosslinker. This effectis described more in detail below.

Surprisingly, it was found that by using sodium acetate buffer with theprotein/polypeptide solution and sodium citrate buffer with thecrosslinker solution in a single composition, the benefits of using eachtype of buffer (ie accelerated crosslinking and improved mechanicalproperties) could be simultaneously achieved, without any inappropriateinhibition of any reaction or any inappropriate additional reactionoccurring. The successful implementation of this approach is describedwith regard to the below illustrative, non-limiting examples.

Buffer pH

PCT Pub. No. WO/2008/076407, filed on Dec. 17, 2007, further disclosesthat according to a preferred embodiment, the pH of the buffer isadjusted to a value in the range of from about 1.5 to about 5.0, or fromabout 7.0 to about 9.0, which is outside the general range of isoionicpH values for gelatins, in order to increase the solubility of gelatinin the solution. According to the teachings of the PCT application, thefurther the product pH is from the isoionic pH the better will be thesolubility of the gelatin. As noted in greater detail below, it has beenfound that for transglutaminase, pH values in a range around pH 6.0 aremost effective.

However, according to accepted practice for compositions that are to beused within living organisms, gelatin should be dissolved in an aqueoussolvent buffered at pH 5.5-9.0, a range that encompasses the pH range ofisotonic gelatin solution.

According to some embodiments of the present invention, the pH of thegelatin solution is optionally and preferably adjusted to fall withinthe range of pH levels at which mTG retains upwards of 80% of itsenzymatic activity. For currently known strains of mTG, this range isfrom about 5 to about 8 (Activa® General Information, Ajinomoto FoodIngredients LLC).

The lower part of the high mTG activity pH range is preferred, since themTG-facilitated cross-linking of gelatin releases NH₃ as a product,which raises the pH in the local environment during a cross-linkingprocess. Without wishing to be limited by a single hypothesis, if theinitial solution pH is at the lower part of the high mTG activity pHrange, then the release of NH₃ during protein cross-linking will notimmediately increase the local pH above the pH range for high mTGactivity, enabling the mTG to function continuously at a high activitylevel. Hence, preferably, the pH of the gelatin solution component isadjusted to a pH in the range of from about 5 to about 8, and morepreferably from about 6 to about 7. Most preferably, the pH of thegelatin solution component is adjusted to a pH of about 6, which is theoptimal pH for activity of crude transglutaminase.

The pH can be adjusted using a pH adjusting agent or any other methodknown in the art. For example and without any intention of beinglimiting, one such method involves titration of a solution of dissolvedbuffer salts with acetic acid glacial until the solution pH reaches thedesired pH level. After pH adjustment, the solution is transferred to avolumetric bottle and double distilled water is added until the desiredbuffer solution volume, and corresponding buffer concentration, isachieved.

Addition of Calcium and Urea

PCT Pub. No. WO/2008/076407, filed on Dec. 17, 2007, discloses that theaddition of calcium chloride (CaCl₂) to gelatin solutions substantiallyreduces the transition point of gelatin. A 25% (w/w) gelatin solution ina buffer of 2M calcium chloride has a transition point below 22° C.,sufficiently low to remain liquid at operating room temperature.However, mTG-facilitated cross-linking of gelatin is greatly inhibitedin such a high CaCl₂ environment, greatly increasing the time requiredfor gelatin gels to cure. Furthermore, the cross-linked gelatin productthat results from mTG cross-linking in such an environment has inferiormechanical properties to cross-linked gelatin product resulting from mTGcross-linking in a lower CaCl₂ environment.

Surprisingly, the present inventors found that calcium chloride and ureahave a synergistic effect on lowering the transition point of gelatinsolutions. When both calcium chloride and urea are incorporated in abuffer solution at appropriate concentrations and gelatin (25% w/w, typeA 300 bloom porcine) is dissolved into that buffer solution, the gelatinsolution transition point can be reduced to below operating roomtemperatures (18-22° C.). In this synergistic solution, the requiredconcentrations of urea and calcium chloride are each much lower than theconcentrations which would be required if only one of these substanceswere used to lower the transition point of a gelatin solution. Forexample, in place of dissolving gelatin in a 2M solution of CaCl₂, theequivalent transition point lowering can be accomplished by dissolvinggelatin in a 1M solution of CaCl₂ that includes 2M of Urea or a 0.5Msolution of CaCl₂ that includes 3M of urea. Experimental examples ofthis effect are described with regard to the below illustrative,non-limiting examples.

In the absence of CaCl₂, a 4-4.5 M solution of Urea is required to lowerthe gelatin solution transition point equivalently. In many cases, whenthis synergistic solution for lowering transition point is used, themechanical properties (cohesive strength, adhesive strength, andelasticity) of cross-linked gelatin gels resulting from mTGcross-linking are improved.

In another embodiment of the present invention, as an alternative to orin addition to CaCl₂, a different calcium compound is used. Examples ofother calcium compounds are calcium hydroxide and calcium carbonate. Theuse of calcium hydroxide to lower the transition point of a gelatinsolution that is then crosslinked using mTG is described with regard tothe below illustrative, non-limiting examples.

In another embodiment, as an alternative to or in addition to a calciumcompound, a compound that incorporates a different divalent cation isused. A non-limiting example of an appropriate divalent compound ismagnesium chloride.

Transglutaminase Concentration

Addition of calcium chloride and/or urea to gelatin solution in order toreduce the transition point of the solution, has an inhibitory effect onmTG-facilitated cross-linking, even in the lower concentrationsdescribed above, thereby slowing the curing of the thermallyirreversible gelatin gelation that is desirable for hemostatic, tissuesealing, tissue adhesion, and other wound treatment applications. Theincrease in mTG-facilitated protein cross-linking time resulting fromthe addition of urea or CaCl₂ may reduce the utility of proteinmTG-crosslinking for the above-mentioned medical applications.

The present inventors have found that the rate of mTG-facilitatedprotein cross-linking reaction speed can be increased by increasing theconcentration of mTG in the mTG solution that is mixed with a proteinsolution to start the mTG cross-linking reaction. Materials and Methodsfor increasing the concentration of mTG in a solution, according to someembodiments of the present invention, include the use of a commerciallyavailable concentrated mTG mixture, the purification of mTG solutionsfrom bulking agents, and the concentration of mTG solutions usingfiltration techniques. Through the use of these methods and materials,the protein concentration of mTG in a mTG solution can be increased upto a concentration of about 2% w/w (20% w/w solution of a 10% by weightmTG product, such as ACTIVA TG).

According to some embodiments, when urea and/or CaCl₂ are added to thegelatin solution buffer to reduce the transition point of gelatinsolutions, the preferred protein concentration of the corresponding mTGsolution is in the range of from about 0.1% to about 2% w/w of total mTGsolution. More preferably, the concentration of the mTG solution is inthe range of from about 0.25% to about 1% w/w of total mTG solution.Most preferably, the concentration of the mTG solution is in the rangeof from about 0.4% to about 0.8% w/w of total mTG solution.

According to some embodiments of the present invention, mTG solution isthoroughly mixed with the gelatin solution at a volume ratio rangingfrom 1:0.5 to 1:8, mTG solution:gelatin solution. Preferably, the volumeratio ranges from 1:1 to 1:4, mTG solution:gelatin solution; morepreferably, the volume ratio ranges from 1:1 to 1:2, mTGsolution:gelatin solution.

These volumetric ratios and mTG concentrations taken together describeembodiments of the current invention wherein the protein concentrationof mTG in the total composition is in the range from about 0.01% toabout 1.35% w/w, preferably in the range from about 0.05% to about 0.5%w/w, and more preferably in the range from about 0.1% to about 0.4% w/w.

According to some embodiments of the invention, the enzyme activity inthe total composition is in the range from about 1 to about 180 enzymeunits (EU) per gram, preferably in the range from about 4 to about 70EU/g, and more preferably in the range from about 10 to about 55 EU/g.

Calcium Sequestering Agents

As discussed above, although CaCl₂ is very useful in lowering thetransition point of solutions comprising cross-linkable proteins orpolypeptides, its presence in a cross-linked gelatin solution hasdeleterious effects. As such, a method of neutralizing the effect ofCaCl₂ once cross-linking has occurred would be extremely useful in thedevelopment of a composition for use in medical and surgical sealant andhemostasis applications.

According to some embodiments of the present invention, the hemostaticor fluid sealing composition comprises calcium-binding agents. When asolution comprising the cross-linking material and at least onecalcium-binding agent is mixed into a solution of a cross-linkableprotein containing CaCl₂, the calcium-binding agents attach to thecalcium molecules and reduce their negative effect on hemostatic orfluid sealing composition. The term “calcium-binding agents” in thecontext of this invention is synonymous with the terms“calcium-sequestering agents,” “calcium-chelating agents,”“calcium-chelators,” “calcium-complexing agents.”

It has further been surprisingly found by the inventors of the presentinvention that the addition of calcium-binding agents to a solution of across-linkable protein, such as gelatin, has additional effects asidefrom neutralizing calcium in the solution. When such agents are added toa gelatin solution, the gelatin solution undergoes physicalthermo-reversible gelation. The rate of this gelation process isdependent on the concentration of the calcium-binding agent in thesolution being added to the gelatin solution. Preferably, theconcentration of calcium-binding agent in an mTG solution is in therange of from about 0.1 to about 0.5M. More preferably, theconcentration range is from about 0.25 to about 0.5M. Above aconcentration of 0.5M, the thermo-reversible gelation that isfacilitated by certain calcium-binding agents may occur rapidly enoughso as to severely hamper the mTG-facilitated cross-linking.

The thermo-reversible gelation triggered by the addition of a solutioncontaining calcium-binding agents to a gelatin solution occurs even withgelatin solutions that do not form thermo-reversible gels at ambienttemperatures. These types of gelatin solutions include those describedabove that contain amounts of urea and/or CaCl₂ that reduce the gelatinsolution transition point below 18-22° C., sufficiently low to remainliquid at operating room temperature.

The thermo-reversible gelatin gels formed by the addition ofcalcium-binding agents to a gelatin solution are distinctly differentfrom the thermo-reversible gelatin gels formed when a gelatin solutionis brought to a temperature below its sol-gel transition point. Thegelation process triggered by a reduction in temperature is generally agradual process since gelatin solutions have low thermal conductivityand it takes a significant amount of time for the temperature of anentire gelatin solution to drop below its sol-gel transition point. Thegel that is formed from the gelatin solution is clear and firm. Thegelation process trigged by calcium-binding agents will occur nearlyimmediately if a sufficient amount of agent is added. The gel that isformed is opaque, white in color, and very stretchy. Furthermore, gelsthat result from calcium-binding agent gelation only revert back intosolution at temperatures above 40° C., their approximate sol-geltransition point, which is significantly higher than the sol-geltransition point of gels that result from of 300 bloom gelatin.

Without wishing to be limited by a single hypothesis, the mechanism forthe thermo-reversible, physical gelation triggered by the addition ofcertain calcium sequestering agents, such as citrate, EDTA and Calgon,possibly results from ionic crosslinking as these agents are negativelycharged polyanions, Type A gelatin has an isoelectric point of 7-9 whichmeans that it is positively charged in solution at pH 6.0 (as in thebelow referenced example). A negatively charged polyanion added to anegatively charged gelatin solution can cause ionic crosslinking.

In another optional embodiment of the present invention, a negativelycharged polyanion that is not a calcium sequestering agent is usedadditionally or alternatively to the calcium sequestering agent oragents to introduce ionic crosslinking to the composition.

The thermo-reversible gelation that can be triggered through theaddition of calcium-binding agents to a solution of gelatin or a relatedcross-linkable protein, has great benefit in improving compositions ofmTG-facilitated cross-linked protein gels for medical and surgicalapplications. This is particularly true with regard to cross-linked gelsmade from gelatin solutions with sol-gel transition points below 18-22°C., which are sufficiently low to remain liquid at operating roomtemperature, such as those that contain the necessary amounts of ureaand/or CaCl₂. The elasticity and cohesiveness of cross-linked gels madefrom gelatin solutions containing transition point-lowering additivesare reduced since the gels then lack the physical, thermo-reversiblegelation that normally takes place in gelatin solutions that makesgelatin gels elastic and cohesive. Reduced elasticity and cohesivenesssignificantly harm these gels when they are used for tissue sealant,hemostatic, or wound closure applications since the gels are then lessable to withstand the flow of blood or other body liquids withoutcracking and permitting the liquid to break through the gel once it isin place on top of the wound site. However, if a calcium-binding agentis added to the mTG solution that is mixed with the gelatin solution tofacilitate cross-linking, then the gelatin solution will also undergophysical, thermo-reversible gelation at the same time that it isundergoing mTG-facilitated cross-linking. The concentration ofcalcium-binding agent useful for this embodiment of this invention isoptionally and preferably an amount that, on its own, will not result inthermo-reversible gelation within the period required formTG-facilitated cross-linking to occur. If too much calcium-bindingagent is added, then thermo-reversible gelation will occur immediatelyand mTG-facilitated cross-linking will not occur at all.

The thermo-reversible gelation triggered by calcium-binding agentsoccurs both in gelatin solutions where calcium or a calcium-containingmolecule is included as part of the solution and in gelatin solutionsthat contain no calcium or calcium-containing molecules. In gelatinsolutions not containing any calcium, the addition of calcium-bindingagents is useful only in the thermo-reversible gelation effect that istriggered. In gelatin solutions that do contain calcium, either alone orin larger molecules, calcium binding agents are useful both in reducingthe deleterious effects of calcium and in triggering thethermo-reversible gelation effect. The deleterious effects of calcium ona crosslinked protein/polypeptide composition can include inferiormechanical properties, specifically increased brittleness and areduction in cohesive strength. Additionally, when calcium is present athigh concentrations above its toxicity threshold, it can result in anadverse tissue response.

Non-limiting examples of calcium-binding agents that are useful in thecontext of the present invention include polyphosphate salts, such aspyrophosphates (including tetrasodium pyrophosphate, disodium dihydrogenpyrophosphate, tetrapotassium pyrophosphate, dipotassium dihydrogenpyrophosphate, and dipotassium disodium pyrophosphate),tripolyphosphates (including pentasodium tripolyphosphate, andpentapotassium tripolyphosphate), higher polyphosphate salts such assodium and potassium tetraphosphates, and hexametaphosphate salts, alsoknown as ‘glassy phosphates’ or ‘polypyrophosphates’, and carboxylates,(such as alkali metal citrate salts, alkali metal acetate, lactate,tartrate and malate salts, alkali metal salts ofethylenediaminetetraacetic acid (EDTA), and editronic acid).

Preferred examples of suitable calcium-binding agents include EDTA andsodium citrate.

An experimental example that described mTG-facilitated cross-linking ofgelatin solutions where EDTA or sodium citrate has been added to mTGsolutions in various concentrations is described with regard to thebelow illustrative, non-limiting examples.

A preferred example of a polyphosphate salt which is useful in thecontext of the present invention is powdered sodium hexametaphosphate(SHMP), sold under the commercial name Calgon™. Calgon forms complexeswith ambient calcium ions in water. When used as part of the presentinvention, Calgon had a similar effect to that of other calcium-bindingagents when introduced into gelatin solutions containing calcium. Whenintroduced into gelatin solutions that did not contain calcium, Calgontriggered a similar thermo-reversible gelation process as othercalcium-binding agents but also had the additional benefit of reducingthe mTG-facilitated cross-linking time of the gelatin gels. Gels createdthat included Calgon were also observed to be more adhesive than gelscreated that included other calcium-binding agents.

Urea-Sequestering and Urea-Hydrolyzing Agents

As discussed above, urea may be added to a gelatin solution so as tolower its transition point significantly. This is of great benefit insimplifying the use of a gelatin-mTG compound in an operating roomenvironment. However, the cross-linked gelatin gels formed in thepresence of high concentrations of urea are non-ideal for certainapplications as they have high osmotic pressure and can draw water outof surrounding tissues when implanted onto native tissue in the body.

According to some embodiments of the present invention, the effect ofurea in a gelatin solution is neutralized, for example by including aurea-complexing agent or a urea-sequestering agent, along with anactivator if necessary, in a mTG solution that is mixed with a gelatinsolution to form a gelatin-mTG composition. Additionally, an agent thatcatalyzes the hydrolysis of urea can be included in the mTG solutionwith similar effect. When the mTG solution is mixed into a gelatinsolution, the urea-sequestering or urea-hydrolyzing agent immediatelyforms a reaction with the urea in the gelatin solution and neutralizes,or reduces, its potentially undesirable effect on the gelatin-mTGcomposition. The urea-sequestering or urea-hydrolyzing agent mayoptionally have a positive effect on the mechanical properties of thegelatin-mTG composition and increase the composition's adhesivestrength, cohesive strength, and/or elasticity (without wishing toprovide a closed list and also without wishing to be limited by a singlehypothesis).

A non-limiting example of an agent that is useful for catalyzing thehydrolysis of urea is urease, a commercially-available enzyme thatcatalyzes the hydrolysis of urea into carbon dioxide and ammonia. Ureaseoccurs in many bacteria, several species of yeast and a number of higherplants. Two illustrative sources are: Jack beans (Canavlia ensiformis)from which it has been crystallized and thoroughly studied, and Bacilluspasteurii.

Urease is preferentially included in an mTG solution at concentrationsranging from about 0.1 M, to about twice the molar concentration of ureain the corresponding gelatin solution to which the mTG solution will beadded to form a gelatin-mTG composition, up to the saturation point ofurease in solution. For example, if 1M of urea is included in a gelatinbuffer, then the maximum preferred concentration of urease is 2M. Thispreferred concentration relates to the 1:2, mTG solution:gelatinsolution ratio that is preferential in the herein described gelatin-mTGcompositions and allows for one molecule of urease for every molecule ofurea.

According to some embodiments of the present invention, urease isincluded in an mTG solution along with a urease stabilizer. Non-limitingexamples of such stabilizers are EDTA, in concentrations of from about1M to about 3 M, or glycerol, in concentrations of approximately 50% v/vin mTG buffer solution. Further examples include glutathione andcitrate, as described in U.S. Pat. No. 4,188,465.

A non-limiting example of a urea-complexing agent is paraffin, which cancomplex urea in the presence of an activating agent, such as lowmolecular weight alcohols and ketones. Such a process is described inU.S. Pat. No. 2,719,145.

Plasticizer

Some embodiments of the present invention comprise the addition ofsorbitol to the solution of the cross-linking material, rather than tothe cross-linkable protein component, of the composition.

Surprisingly, as described in greater detail below, it has been foundthat sorbitol included in a mTG solution can unexpectedly combine with agelatin solution prior to the onset of gelatin cross-linking. Sorbitol,when added to the mTG solution prior to mixing with the gelatinsolution, increases the flexibility and elasticity of gelatin-mTGcompositions. Such increased flexibility and elasticity may optionallyrepresent improved properties for certain applications or uses of thecompositions. Furthermore, sorbitol can act as a carrier molecule forthe mTG, protecting it from oxidation and increasing the shelf-life ofmTG solutions.

Though included in the mTG solution, the preferred concentrations ofsorbitol to be added are preferably determined with reference to theamount of gelatin in the gelatin solution that corresponds to aparticular mTG solution. The preferred concentration of plasticizer,expressed as a weight ratio of the amount of gelatin in thecorresponding gelatin solution, ranges from 1:1 to 3:1. The amount ofplasticizer added to the solution within this range does not have asignificant effect on the cross-linking time of a gelatin solution.

As discussed above, the properties of an mTG-facilitated cross-linkedgelatin composition can be altered depending on the buffer used for themTG solution. For example, when sodium citrate buffer is used, thesorbitol-containing gelatin-mTG composition is extremely flexible. Whensodium acetate buffer is used, the cross-linking time required to form avigorous gelatin gel is greatly reduced. Both with sodium citrate andwith sodium acetate buffer solutions, the gelatin-mTG composition is farmore elastic and flexible in the presence of sorbitol than it is in theabsence of sorbitol.

The addition of sorbitol to mTG solutions made with sodium citratebuffer described with regard to the below illustrative, non-limitingexamples. The experimental data in this experimental example confirmsthat sorbitol further enhances the flexibility of gels made with mTG insodium citrate buffer.

mTG facilitated cross-linking of gelatin gels, when sorbitol is includedin the mTG solution, at a more rapid rate when sodium acetate is used asthe buffer for the mTG solution in comparison to gels made when sodiumcitrate is used as the buffer, as described in greater detail below.

Further examples of plasticizers which may be used in the context of thepresent invention include, without limitation, citric acid alkyl esters,glycerol, glycerol esters, phthalic acid alkyl esters, sebacic acidalkyl esters, sucrose esters, sorbitan esters, acetylatedmonoglycerides, glycerols, fatty acid esters, glycols, propylene glycol,lauric acid, sucrose, glyceryl triacetate, poloxamers, diethylphthalate, mono- and di-glycerides of edible fats or oils, dibutylphthalate, dibutyl sebacate, polysorbate, polyethylene glycols (PEG) 200to 20,000, Carbowax polyethylene glycols, polyvinyl alcohol (PVA), gumarabic, guar gum, xanthan gum, Plasdone® (polyvinylpyrrolidone),mannitol, and mixtures thereof.

Gum arabic, guar gum, PVA, PEG 6000, and Plasdone were shown to increasethe flexibility of mTG-crosslinked gelatin composition. Preferably, theplasticizer of the present invention comprises one or more of sorbitol,polyethylene glycol, polyvinyl alcohol, or gum arabic, although otheruseful plasticizers are also encompassed within these embodiments of thepresent invention as described herein.

Kosmotropes and/or Osmolytes

Kosmotropes are substances that increase the order of water molecules inthe solvation layer around proteins in aqueous solution and as a result:

-   -   Stabilize macromolecules and proteins in aqueous solutions    -   Increase hydrophobic effects, intermolecular interactions, and        aggregation of proteins.

The effect of kosmotropes on biomolecules is usually opposite to that ofchaotropes, such as urea and guanidinium chloride (GuCl). Chaotropesdisrupt the structure of water and, as a result, new hydrogen bonds areformed between water and the protein at the expense of protein-proteininteractions resulting in:

-   -   Solubilization of aggregates    -   Unfolding of globular proteins by exposing internal hydrophobic        regions in the proteins to the solution

As described above, chaotropes such as urea and GuCl can be used todisrupt the physical gelation of certain protein/polypeptide solutionsby destabilizing the hydrogen bonding network between theprotein/polypeptide chains. These chaotropes thus lower the sol to geltransition temperature of these solutions.

However, disrupting the physical gelation of protein/polypeptidesolutions can, in some circumstances, have adverse effects on themechanical properties of a crosslinked protein composition. For example,physical gelation can act to increase the elasticity of suchcompositions. Disrupting the physical gelation can then reduce theelasticity of these compositions and increase their brittleness, whichmay be undesirable for certain applications.

By incorporating a kosmotrope into the crosslinking material solution,physical gelation can be stimulated to occur in combination with,preferably simultaneously to, the gelation mediated by the crosslinkingmaterial, thus maintaining the beneficial mechanical property effects ofphysical gelation.

In some embodiments of the present invention, the sol-gel transitionpoint of the protein/polypeptide solution has been lowered and one ormore kosmotropes are incorporated into the crosslinking material orcrosslinking material solution.

In a preferred embodiment, the kosmotrope is added at a concentrationsufficient to cause physical gelation in a protein/polypeptide solution.

A non-limiting range of kosmotrope concentrations is 0.5-1M of thecombined crosslinked protein composition.

In some embodiments, the kosmotrope is an ionic kosmotrope.

In a preferred embodiment, the kosmotrope is a non-ionic kosmotrope.

Non-limiting examples of non-ionic kosmotropes are proline andtrehalose.

Non-limiting examples of ionic kosmotropes are trimethylamine N-oxide(TMAO) and glutamate (glutamic acid).

The stabilizing effect of kosmotropes on protein stability in vitro andtheir counteraction of urea may be related to their in vivo function asosmolytes. A good correlation between these in vitro and in vivofunctions has been demonstrated for proline (Fisher M T et al., PNAS103, 2006: p. 13265-6).

In a preferred embodiment, the kosmotrope for use in the presentinvention is an osmolyte.

Non-limiting examples of an osmolyte are glutamate or proline.

Crosslinker Inhibitors

As the crosslinking density in a protein/polypeptide solution isincreased, the stiffness of the crosslinked composition is increased.Therefore, where increased flexibility or elasticity is desired from acrosslinked protein composition, it can be useful to limit thecrosslinking density in the composition. One manner of accomplishingthis is to reduce the amount of crosslinking catalyzed by thecrosslinking material in the protein solution by introducing acrosslinker inhibitor into the composition.

In an embodiment of the present invention, a crosslinking inhibitor isadded to either the protein solution or crosslinker solution such thatthe crosslinking level of the crosslinked composition is reduced.

In a preferred embodiment of the present invention, the crosslinkingmaterial is an enzyme and the inhibitor is an enzymatic inhibitor.

In a more preferred embodiment of the present invention, thecrosslinking material is a transglutaminase (TG) and the inhibitor is atransglutaminase inhibitor.

In a more preferred embodiment of the present invention, thecrosslinking material is a microbial transglutaminase (mTG) and theinhibitor is an mTG inhibitor.

Non-limiting examples of mTG inhibitors include cystamine, an organicdisulfide that can form a disulfide bridge with mTG, cysteine, ahydrophobic amino acid with a reactive S—H side chain (thiol group),melanin, denaturants, other compounds with thiol groups or disulfidebonds, lysine, and compounds <5 kDA in size containing mTG substrates.

Thiol side chains, such as those in cystamine, cysteine, dithiothreitol(DTT), and mercaptoethanol, can serve as inhibitors as they can blockthe active sit of mTG by reacting with the mTG thiol group.

Melanin was previously described as having function as a competitive mTGinhibitor (Ikura K et al. Biosci Biotechnol Biochem. 66(6), 2002, p.1412-1414).

In a preferred embodiment of the present invention, the inhibitor isincluded in quantities sufficient to inhibit crosslinking activity byless than 50%. In a more preferred embodiment, it is included inquantities sufficient to inhibit crosslinking activity by less than 30%.

In another embodiment of the present invention, the inhibitor isreleased into the composition after crosslinking has already begun.

PEG or PVA Copolymers of Crosslinkable Protein/Polypeptide

According to some embodiments, polyethylene glycol (PEG), also known aspoly(ethylene oxide) (PEO) or polyoxyethylene (POE), is added to theprotein/polypeptide or crosslinker solution as a copolymer, to improveone or more properties of the composition, for example (and withoutlimitation) to increase the flexibility of the composition or to shieldfrom the body's immune response to the protein-crosslinker composition.PEG is available over a wide range of molecular weights from 300 Da to10 MDa and may be a liquid or low-melting solid, depending on themolecular weights.

Different forms of chemically-modified PEG are also available, dependingon the initiator used for the polymerization process, the most common ofwhich is a monofunctional methyl ether PEG (methoxypoly(ethyleneglycol)). PEGs are also available with different geometries. BranchedPEGs have 3 to 10 PEG chains emanating from a central core group. StarPEGs have 10-100 PEG chains emanating from a central core group. CombPEGs have multiple PEG chains normally grafted to a polymer backbone.All of these types of PEGs should be considered useful in the presentinvention.

PEGs can be added to either the protein or crosslinker components of aprotein-crosslinker composition. Preferentially, PEG is added at a dryweight ratio between 20:1 to 1:1, protein:PEG. PEG can be added to theprotein component or crosslinker component through modification of theprotein or crosslinker and/or modification of the PEG molecules. Oneexample of such modification is the process known as PEGylation.PEGylation is the act of covalently coupling a PEG structure to anotherlarger molecule. This process can be performed on either the protein orcrosslinker molecules.

The gelatin PEGylation embodiment has, among its many advantages andwithout wishing to be limiting, the advantage that the PEG is part ofthe protein chain, therefore inducing changes in properties of theprotein surface including but not limited to charge and hydrophilicity,as well as steric effects that are due to its bulkiness. As a result,the covalently attached PEG can have profound effects on intermolecularinteractions between protein chains and in turn on physical gelation andcrosslinker dependent crosslinking as well as on the mechanicalproperties of gels prepared by these methods.

The PEG molecules used in PEGylation are usually activated, meaning theyreact spontaneously with functional groups on the target protein. A nonlimiting example of PEGylation is using NHS ester derivatives of PEG.These activated PEG molecules react with primary amines on proteins toform amide bonds with the release of N-hydroxy-succinimide (NHS).

Other ways in which a protein can be modified is by reacting the primaryamines found inside chains of lysine and at the amino termini of theprotein chains. The modification may be by alkylation, succinylation,carbamylation, or by any other method of protein modification.

In a preferred embodiment, the crosslinkable protein/polypeptide isfirst reacted with activated PEG to create PEGylated protein. ThePEGylated protein is purified from excess unreacted PEG and otherreaction products by methods such as, but not limited to, dialysis,ultrafiltration, and gel filtration chromatography. The PEGylatedprotein can then be reacted with a crosslinker to form a crosslinkedgel,

PEGs can also optionally be added through the use of PEG amine as asubstrate for a crosslinker that targets amine groups. The crosslinkercrosslinks the PEG molecule through its terminal amine group tocrosslinker substrates on the protein molecule, thus competing with thenatural amine groups on the protein.

PEG amines comprise PEG that has been bound to amine-functional groups.These are commercially available in all types of PEG geometries. Sourcesof amine-functional PEG products include NOF (Japan), Nanocs (New York,N.Y.) and Pierce Biotechnology (Rockford, Ill.).

In all approaches of incorporating PEG, the number of natural substratesavailable for crosslinking is reduced, resulting in reducedcross-linking. This may affect the mechanical properties of thecrosslinked gel, for example optionally allowing it to become less rigidand more flexible. In addition and without wishing to be limited by asingle hypothesis, the PEG molecule itself may act as a plasticizer andfurther contribute to the flexibility of the resulting gel.

According to a preferred embodiment, the PEG amine comprises activelysine amino acids.

According to another embodiment, of the present invention, PolyvinylAlcohol (PVA) is added to a gelatin or mTG solution as a copolymer toincrease the flexibility or adhesiveness of a protein-crosslinkercomposition. PVA is a water-soluble synthetic polymer with high tensilestrength and flexibility. In a high humidity environment, such as insidethe body, PVA will absorb water. The water, which acts as a plasticizer,can then reduce the tensile strength of the PVA, but increase itselongation.

According to some embodiments, the copolymer comprises PVA-amine. Whenthe amine-targeting crosslinker is added to the solution, both theprotein and PVA-amine will act as substrates and a protein-PVA copolymerwill be formed with better flexibility than a comparable cross-linkedprotein polymer.

A non-limiting example of a process that can be used for producing aminefunctional derivatives of poly (vinyl alcohol) is described in U.S. Pat.No. 6,107,401.

Another non-limiting example of a process that can be used for producingan amine copolymer of PVA is described in U.S. Pat. No. 4,931,501 wherepoly(vinyl alcohol) is reacted with an amino-aldehyde dialkyl acetal.

A process of synthesizing amine-modified poly(vinyl alcohol)s by atwo-step process using carbonyl diimidazole activated diamines toproduce PVAs with different degrees of amine substitution has alsopreviously been described (Wittman M, et al. Biophysical andTransfection Studies of an Amine-Modified Poly(vinyl alcohol) for GeneDelivery. Bioconjugate Chem., 16 (6), 1390-1398, 2005), as anothernon-limiting example.

Surfactants

According to some embodiments of the present invention, one or morebiocompatible surfactants are added to the solution of cross-linkableprotein or polypeptide, for example in order to reduce the surfacetension of that solution.

Surfactants are wetting agents that lower the surface tension of aliquid, allowing easier spreading, and lower the interfacial tensionbetween two liquids. Lower surface tension facilitates easier handlingof a solution of a cross-linkable peptide as it is easier to passthrough an applicator, and easier to mix with a solution of across-linking material. Surfactants can also lower the viscosity of thesolution. Additionally, lowering the surface tension of a gelatinsolution has great utility when a gelatin solution is lyophilized eitheralone or together with a mTG solution, as it can prevent the formationof a film on the top layer of the dried gelatin. Such a film inhibitsthe reconstitution of lyophilized gelatin into a homogenous solution.

Non-limiting examples of biocompatible surfactants useful in context ofthe present invention are polysorbate 20 (Tween™ 20),polyoxyethyleneglycol dodecyl ether (Brij™ 35),polyoxyethylene-polyoxypropylene block copolymer (Pluronic™ F-68),sodium lauryl sulfate (SLS) or sodium dodecyl sulfate (SDS), sodiumlaureth sulfate or sodium lauryl ether sulfate (SLES), poloxamers orpoloxamines, alkyl polyglucosides, fatty alcohols, fatty acid salts,cocamide monoethanolamine, and cocamide diethanolamine.

Surfactants may be used also as plasticizers. Tween80 for example hasbeen shown to reduce the glass transition point (T_(g)) of severalhydrophilic polymers. The presence of the smaller molecules of Tween80within the polymer were thought to dilute and weaken the cohesiveinteractions between the polymers chains. This reduced the friction andentanglement by increasing the free volume in the polymer matrix.(Ghebremeskel et al, 2006, International Journal of Pharmaceutics328:119-129).

In a preferred embodiment of the present invention, one or moresurfactants are used as a plasticizer to improve the elasticity of thecrosslinked composition, particularly as it stiffens over time.

In another optional embodiment, one or more surfactants are combinedwith another plasticizer from the plasticizers listed above as relevantto the present invention. Rodriguez et al (Food Research International39 (2006) 840-6) demonstrated a synergistic effect between a plasticizer(glycerol) and surfactants (Tween20, Span 80, Lecithin) on increasingthe elasticity of non-crosslinked dry gelatin films.

Preferentially, surfactants are added to a gelatin solution at a weightratio of 0.1-5% of the dry weight of gelatin in the solution.Alternatively, surfactants are added to a gelatin solution at aconcentration approximately equal to the critical micelle concentration(CMC) of that particular surfactant in solution. The CMC of eachsurfactant varies and is dependant on the ionic concentration of thesolution into which the surfactant is dissolved.

Tissue Substrate Neutralization

According to some embodiments of the present invention, the hemostaticor body fluid sealing composition further comprises substrate-specificbinding agents, which neutralize adhesion-inhibiting effects of surfacesubstances on a tissue substrate being sealed, attached, or otherwisetreated, with the composition. For example, these agents can bind,dissolve, and/or disperse substrate surface substances withanti-adhesive effects.

According to a preferred embodiment, the composition of the presentinvention is targeted to adhere to mucous membranes, such as that of thegastrointestinal tract (GIT) or the buccal mucosa.

Tissues in the GIT, such as the intestines, are frequently mucosal,covered with mucus that is continuously produced. Since the mucus isconstantly refreshing itself, successful adhesion to tissue in the GITrequires the composition to specifically adhere to the tissue itself,under the mucosal layer.

One way of specifically adhering targeting regions of the GIT is byusing mucoadhesives that can reversibly bind to cell surfaces in theGIT. These mucoadhesives function with greater specificity because theyare based on receptor-ligand-like interactions in which the moleculesbind strongly and rapidly directly onto the mucosal cell surface ratherthan the mucus itself.

A non-limiting example of a class of compounds that has these uniquerequirements are lectins. Lectins are proteins or glycoproteins andshare the common ability to bind specifically and reversibly tocarbohydrates. They exist in either soluble or cell-associated forms andpossess carbohydrate-selective and recognizing parts. The intestinalepithelial cells possess a cell surface composed of membrane-anchoredglycoconjugates. It is these surfaces that could be targeted by lectins,thus enabling an intestinal delivery concept (Shah K U, Rocca J G., DrugDeliv. Tech., 2004, 4(5), 1).

A non-limiting example of a mucoadhesive lectin is tomato lectin (TL).TL has been extensively studied in in vitro binding and shown to bindselectively to the small intestine epithelium (Lehr C, Bouwstra J A, KokW, Noach A B, de Boer A G, Junginger H E. Pharma Res., 1992, 9(4),547-53.)

Lectins are useful for improving adhesion to all mucosal tissue surfacesthat contain membrane-anchored glycoconjugates. Non-limiting examples ofsuch tissue surfaces are buccual mucosa and the walls of the intestinaltract.

A non-limiting example of a compound to improve buccal mucoadhesion is acopolymer of poly(aspartic acid) (PAA) and polyethylene glycol (PEG)monoethylether monomethacrylate (PAA-co-PEG) (PEGMM) (Shojaei A M, Li X.J. Control. Release, 1997, 47, 151-61.27.) Preferably the PEGMM is16-mole % PEGMM, which has the most favorable thermodynamic profile andthe highest mucoadhesive forces.

According to some embodiments of the present invention, the mucoadhesiveis a commercially available mucoadhesive hydrogel. Non-limiting examplesof hydrogels suitable for use in improving the substrate specificadhesion of the herein described novel compositions are available underthe tradename Corplex® (Corium Technologies, Menlo Park, Calif.). Theseadhesive hydrogels are prepared by non-covalent (hydrogen bond)cross-linking of a film-forming hydrophilic polymer (for examplepolyvinyl pyrrolidone, (PVP)) with a short-chain plasticizer (typicallybut not necessarily PEG) bearing complementary reactive hydroxyl groupsat its chain ends.

Another non-limiting example of mucoadhesive compounds includes polymerswith one or more thiol groups. In such polymers, the introduction of asulphahydryl group increases the adhesive properties of the mucoadhesivepolymers (Bernkop-Schnurch A, Schwarch V, Steininger S. Pharm. Res.,1999, 16, 6, 876-81.32). The improved adhesion was demonstrated onporcine intestinal mucosa. Thiolated polymers (thiomers) have alsodemonstrated strong buccal adhesive properties (Langoth N, Kalbe J,Bernkop-Schnurch A. Int. J. Pharm., 2003, 252, 141-48.)

Another non-limiting example of a buccal adhesive is a naturalmucoadhesive gum derived from Hakea (Alur H H, Pather S I, Mitra A K,Johnston T P. Int. J. Pharm., 1999, 88(1), 1-10.)

Enzyme Purification & Concentration

According to some embodiments of the present invention, transglutaminasesolutions undergo one-stage or multiple-stage purification to performone or more of 1) remove fermentation residue from the transglutaminasemixture; 2) concentrate the amount of active transglutaminase in atransglutaminase solution; 3) further purify the transglutaminasesolution from carrier proteins or carbohydrates; 4) lower the endotoxinlevel of the transglutaminase solution; and/or 5) remove all microbesfrom the transglutaminase solution, effectively sterilizing thesolution; all without wishing to be limited to a closed list.

The present invention, in at least some embodiments, further provides amethod for preparing a hemostatic or body fluid sealing composition, themethod comprising providing a solution of a cross-linkable protein orpolypeptide; providing a solution of a cross-linking material; andmixing the solution of the cross-linkable protein or polypeptide withthe solution of cross-linking material.

According to some embodiments, the solution of cross-linking material isfiltered prior to mixing with the cross-linkable protein of polypeptide.

In a preferred embodiment of this invention, the filtration processfirst uses coarse filtration, sometimes known as clarification, toremove large blocks of fermentation residue that will rapidly blockfiner filtration steps. Non-limiting examples of such coarse filtrationis about 0.45 μm pore size filtration and about 0.65 μm pore sizefiltration.

According to another preferred embodiment of the present invention, thesolution of cross-linking material is optionally and preferably passedthrough a filter of pore size of below 0.22 μm, for example to reducethe bioburden of the material below 10 colony forming units (CFU) pergram and make it appropriate for medical use. Preferably, the bioburdenis practically eliminated to achieve a sterility assurance level (SAL)of less than about 10⁻² and more preferably less than about 10⁻³, whereSAL is a term used in microbiology to describe the probability of asingle unit being non-sterile after it has been subjected to asterilization process.

According to another preferred embodiment of the present invention,either tangential flow or hollow fiber ultra-filtration techniques areused, not only to purify the solution of cross-linking material byremoval of carrier carbohydrates and proteins, but also to concentratethe solution. Preferred pore sizes for use with this invention are thosewith pore sizes smaller than the size of the components of thecross-linking composition.

In a preferred embodiment, the crosslinking material is mTG and the poresize is in the range of 10-50 kDa. In a more preferred embodiment, thecrosslinking material is mTG and the pore sizes are in the range of10-30 kDa.

According to another embodiment, on or more size exclusionchromatography steps is used to selectively separate the crosslinkingmaterial from surrounding substances.

According to another embodiment, one or more hydrophobic or hydrophilicinteraction chromatography steps is used to selectively separate thecrosslinking material from surrounding substances.

According to another preferred embodiment of the present invention, thecrosslinking material is a protein and one or more ion exchangechromatography steps is used to preferentially bind the crosslinkingprotein, thereby purifying it from the surrounding materials.

According to a more preferred embodiment, the crosslinking protein ismTG and one or cation exchange chromatography steps is used to purifythe mTG.

In a preferred embodiment, the cation exchange resin is a sepharoseresin.

According to another preferred embodiment, purification reduces theendotoxin level of the crosslinking material to <5 endotoxin units (EU)per gram.

According to another preferred embodiment, the crosslinking material ismTG and purification results in an mTG composition wherein the specificactivity is greater than 20 enzyme units per milligram and preferablygreater than 25 units per milligram.

According to another preferred embodiment, the crosslinking material ismTG and purification results in electrophoretic purity of at least 95%and preferably of at least 98%.

An mTG purification process, as a non-limiting example, is describedherein that purifies a food-grade mTG product to produce an mTGcomposition with specific activity >25 enzyme units per milligram, >95%electrophoretic purity, <5 endotoxin units per gram, and <10 CFU/g.

As described above, mTG concentration is also a preferred parameter forsome embodiments of the composition of the present invention. The abovepurification processes may also result in more concentrated mTGmaterial. In addition to cross-linking gelatin more rapidly thannon-concentrated mTG solutions, concentrated mTG solutions formed gelsthat were more elastic, more adhesive, and more transparent compared tothe non-concentrated controls.

Increased Viscosity of Enzyme Solution

According to another embodiment of the present invention, the viscosityof the non-toxic cross-linker solution is increased so as to decreasethe viscosity disparity between the cross-linker and gelatin solutions.A reduced disparity in solution viscosity enables rapid and homogenousmixing of the two solutions.

In a preferred embodiment of this invention, the viscosity of thecross-linker solution is increased to between 50 and 5000 cP.

In a more preferred embodiment of this invention, the viscosity of thecross-linker solution is increased to between 150 and 2500 cP.

In a non-limiting example of this embodiment, high molecular weightmolecules without amine functionality are added to the cross-linkersolution to increase its viscosity. Non-limiting examples of suchmolecules are soluble starches, polyvinyl alcohols (PVA), polyethyleneglycol (PEG).

In a preferred embodiment of this invention, one or more viscosityincreasing agents is added to the crosslinker solution to increase theviscosity of the solution without inhibiting the crosslinking reactionby more than 50%, preferably without inhibiting the reaction by morethan 30%, and even more preferably without inhibiting the reaction.Non-limiting examples of appropriate viscosity increasing agents includealginate ester, gum arabic, carboxymethyl cellulose (CMC), xanthan gum,guar gum, and plasdone.

Inhibition of Carbamylation in Cross-Linkable Proteins/Polypeptides

As described earlier in the present patent, urea is used in someembodiments of this invention to decrease the sol-gel transition pointof a protein solution. One disadvantage that can arise from the use ofurea is that it can dissociate into cyanic acid. The cyanate ion reactswith primary amine groups on the protein to yield a carbamylatedderivative. This defunctionalizes amine groups on the crosslinkableprotein in a process known as carbamylation. In preferred embodiments ofthe present invention, one of the crosslinking substrates in thecrosslinkable protein comprises one or more amine groups. When thesegroup(s) are defunctionalized, the number of available substrates forcrosslinking are reduced and the crosslinking rate decreases. The timeduring which urea is present in solution as well as the temperature ofthe solution affects the rate of carbamylation, since urea decomposesinto cyanates over time at a rate that is temperature dependant.

It is not surprising that carbamylation is potentially problematic forthe described embodiments of the present invention, as this process haspreviously been described as having adverse effects on amine-functionalproteins and polypeptides. U.S. Pat. No. 4,605,513 describes a methodfor inhibiting carbamylation of polypeptides by using 1,2-ethylenediamine or compounds that are structurally related to 1,2-ethylenediamine. U.S. Pat. No. 7,459,425 B2 describes a process for inhibitingcarbamylation of polypeptides in a urea or cyanate containing solutionby adding a carbamylation inhibitor.

The carbamylation inhibition processes described in the background artrely on the use of competitive amine group substrates to bind thecyanate ions that would otherwise cause carbamylation on the targetmolecule. These background art processes can cause problems with regardto preferred embodiments of the present invention, since thecrosslinkable substrates of the crosslinkable protein include aminegroups. Therefore, the addition of competitive amine group substratescan competitively inhibit the crosslinking of the target crosslinkableprotein. As an example of this, hydroxylamine, which was disclosed as apreferred carbamylation inhibitor in U.S. Pat. No. 7,459,425 B2, wasfound by the present inventors to increase the crosslinking time ofprotein solutions that had been incubated with urea.

The crosslinking material may optionally comprise a chemical entity thathas functional groups which react with primary amines, including but notlimited to aryl azides, Carbodiimide, Hydroxymethyl Phosphine,imidoesters, NHS-esters, vinyl-sulfones, diisocyanates and aldehydessuch as glutaraldehyde and formaldehyde for example.

The cross-linking material may also optionally (alternatively oradditionally) comprise an enzyme crosslinker with an amine groupsubstrate. Preferably, the enzyme crosslinker use the epsilon aminogroups of lysines as a substrate. Non-limiting examples of such anenzyme are microbial transglutaminase (mTG) and tissuetransglutaminases.

It would be expected that the compounds described in the background artwill inhibit transglutaminase dependent crosslinking because thesecompounds contain primary amines which are preferred substrates oftransglutaminases. Surprisingly, it has been discovered by the presentinventors that carbamylation in a protein/polypeptide solution can beinhibited through the addition of a competitive carbamylation inhibitorwithout adversely affecting amine-group dependant crosslinking of theprotein/polypeptide, in contrast to the teachings of the background art.

In an embodiment of the present invention, a carbamylation inhibitor isincluded in the crosslinkable protein/polypeptide composition.

A non-limiting description of a carbamylation inhibitor is any moleculethat can be a more preferable substrate for carbamylation than thetarget protein/polypeptide.

Non-limiting examples of inhibitors are molecules that have primaryamines in their structure.

In a preferred embodiment, the carbamylation inhibitors do not inhibitthe crosslinkability of the polypeptide or do not inhibit itscrosslinkability to a greater extent than they inhibit the carbamylationreaction.

In another preferred embodiment, the carbamylation inhibitors do notinhibit the crosslinker activity or do not inhibit its activity to agreater extent than they inhibit the carbamylation reaction.

According to preferred embodiments, the carbamylation inhibitorcomprises an amino acid or amino acid salt.

According to a more preferred embodiment, the carbamylation inhibitor isone or more of glycine or histidine. According to a more preferredembodiment, the carbamylation inhibitor is glycine.

The glycine is optionally and preferably present at a concentration fromabout 0.05M to about 1.5M in the composition. More preferably, theglycine is used at concentrations from about 0.1 M to about 0.9M.

Surprisingly, it was found by the inventors that although glycinecontains an amine group, it did not inhibit transglutaminasecrosslinking activity or the kinetics of mTG-dependant gelatincrosslinking.

Glycine was shown to inhibit carbamylation preferentially in a gelatinsolution containing urea, as compared with other primary amine groupcontaining substances, and in a dose dependant manner. Histidine canalso optionally be used to inhibit carbamylation.

Sodium cyanate also results in an inhibitory effect, confirming thaturea breakdown is responsible for inhibition of mTG crosslinking.

Modification of Amine Groups in Crosslinkable Protein

In another embodiment of the present invention, the crosslinkableprotein/polypeptide contains amine groups that are a substrate for thecrosslinking material and the primary amine groups on the crosslinkableprotein/polypeptide are modified.

In gelatin, the relevant amine groups are the epsilon amine on lysineside chains and the free amine at the amino termini of the gelatinchains.

Modifying amine groups on the crosslinkable protein/polypeptide candecrease the number of crosslinkable substrates available forcrosslinking, thereby improving the mechanical properties of thecrosslinked protein composition by maintaining the composition'selasticity or changing the chemical properties, such as surface chargeor hydrophibicity, of the composition.

In an embodiment of the present invention, modification of primaryamines may optionally be performed with one or more methods, includingbut not limited to alkylation, amidation, carbamylation, orsuccinylation.

In another embodiment, primary amine groups on gelatin can optionally bemodified by reacting the protein with acetic anhydride, glutaricanhydride and citraconic anhydride.

In another embodiment, primary amines groups may also optionally bemodified by reacting with derivatives of succinimidyl esters. Inaddition, primary amine groups may be modified by PEGylation. In apreferred embodiment, the protein/polypeptide is succinylated.

Succinylation is a process in which succinic anhydride reacts with theε-amino group of lysine and/or the amino-N-terminal α-amino group of aprotein/polypeptide.

In a preferred embodiment, the crosslinking material is atransglutaminase and the primary amine groups on the crosslinkableprotein are modified.

The succinylation of lysines in casein, for example, was shown to renderthe protein a non-substrate for transglutaminase (Nio et al,Agricultural and Biol Chem 50(4), 1986: p. 851-855).

In a preferred embodiment of succyinylation, succinic anhydride isoptionally and preferably added to the protein/polypeptide to start thesuccinylation reaction. When the desired level of succinylation isachieved, the succinylated protein is preferably separated or purifiedfrom excess unreacted succinic anhydride and other reaction products bymethods including but not limited to dialysis, ultrafiltration or gelfiltration chromatography, or a combination thereof. Optionally theexcess unreacted succinic anhydride is chemically reacted with one ormore additives so as to functionally remove it from further reactions.

In another embodiment, the modified protein is optionally and preferablymixed with non-modified protein at ratios ranging from 1:10-10:1 weightmodified protein per weight non-modified protein.

The modified protein may optionally be mixed with the non-modifiedprotein prior to addition of crosslinking material or may optionally beadded to the crosslinking material such that it is mixed with thenon-modified protein only when the crosslinking material is added.

In another embodiment, a protein that would normally be a substrate forthe crosslinking material is preferably modified and used to increasethe viscosity of the crosslinking material solution.

In another embodiment, the crosslinking material is transglutaminase anda protein is preferably modified such that a majority of its lysinegroups are rendered non-functional substrates, leaving glutamine groupsthat can be cross-linked by mTG in the presence of diamine linkers,including but not limited to ethylenediamine, diaminohexane, putrescine,cadaverine, spermine, spermidine and jeffamine.

In a preferred embodiment, the protein is gelatin.

Complete succinylation of gelatin caused gelatin to no longer becrosslinked by mTG.

Also as described below succinylated gelatin may optionally be used in amixture with non-modified gelatin, for example and without limitation toimprove the mechanical properties of a crosslinked gelatin composition.

In another preferred embodiment of the present invention, theprotein/polypeptide in the composition is optionally and preferablymodified through carbamylation.

Carbamylation of a protein is a process in which isocyanic acid reactswith amino group of a peptide and forms a carbamylated peptide.

Carbamylation may optionally be used to modify amine groups when aminegroups of proteins or peptides are used as substrate for crosslinkingprocesses.

In a non-limiting embodiment, cyanate is optionally and preferably addedto the protein/polypeptide to start the carbamylation reaction. When thedesired level of carbamylation is achieved, the carbamylated protein isoptionally separated or purified from excess unreacted cyanate and otherreaction products by one or more methods including but not limited todialysis, ultrafiltration or gel filtration chromatography, or acombination thereof. Optionally the excess unreacted cyanate ischemically reacted with one or more additives so as to functionallyremove it from further reactions.

In a non-limiting embodiment, cyanate is preferably added directly tothe protein/polypeptide before the start of the crosslinking reaction orduring the crosslinking reaction to cause the carbamylation reactions.

Examples of cyanates that can be used for this purpose include but arenot limited to sodium cyanate, ammonium cyanate, and potassium cyanate.

In another embodiment, the protein solution preferably contains urea andcarbamylation begins as urea breaks down into ammonium cyanate.

Partial carbamylation is preferably accomplished by the addition ofcyanates to the total protein-crosslinker composition at a concentrationrange of 0.001-0.1 mM per g protein. Preferably, cyanates are added at aconcentration range of 0.01-0.05 mM per g protein to achieve partialcarbamylation.

Concentrations above 0.05 mM per g protein, and preferably above 0.1 mM,may optionally be used to cause total carbamylation of the amine groupsin a protein.

Cross-Linking Bridge Between Protein Molecular Chains (DiamineMolecules)

According to a further embodiment of the present invention, diamines forwhich transglutaminase has specific activity are added to a protein orcrosslinker composition so as to be incorporated into the crosslinkedprotein composition as improve certain properties of the gelatin-mTGcomposition by creating cross-linking bridges between proteincrosslinks. Non-limiting examples of the benefits of cross-linkingbridges (and without wishing to provide a closed list) include one ormore of increased elasticity, modified bioabsorption time, or modifiedcohesive strength.

As above, this embodiment may optionally be useful for example if thecrosslinkable protein contains amine groups that are a substrate for thecrosslinker material

In some cases, diamines can also be used to inhibit crosslinkingkinetics. If the crosslinker targets epsilon amine groups on lysine sidechains then the diamine can compete with the target substrates of thecrosslinker and slow the crosslinking reaction.

Non-limiting examples for diamines that can be used are putrescine,cadaverine, hexanediamine, spermidine, and spermine. In addition, adiamine of the polyetheramine type, such as Jeffamine EDR-148 (Huntsman)may be used. Lysine and polylysine or a peptide containing 2 or morelysine residues may be used as well.

As mentioned in the modified amine group section, in another embodimentof the present invention, a transglutaminase is the crosslinker and oneor more diamines may optionally be added to a protein solution where theamine groups have been modified. In such cases, the modified protein orsome peptides therein may be not be cross-linkable by themselves. Thediamine then provides primary amines for crosslinking bytransglutaminase to take place. Among the many advantages of thisapproach is that the diamines would not compete with the natural lysinesand slow down the crosslinking reaction. Rather, they would bridge theglutamine substrates in the target protein to each other through thediamine bridges. This process results, without wishing to be limited bya single hypothesis, in gels with longer and more flexible bridges inorder to improve the flexibility of the crosslinked composition.

Some diamines can be used to modify the rate of protease biodegradationof the crosslinked protein composition. For example, lysine andhexanediamine have demonstrated the ability to reduce or accelerate therate of protease biodegradation (Ma et al. Biomaterials. 2004, 25(15):p. 2997-3004).

In a preferred embodiment, the cross linking bridge is formed betweenlysine side chains in the crosslinkable protein/polypeptide through adiamine.

Non-limiting examples of diamines that can be used in this context areadipic diamide and glutaric diamide.

The effect of a diamine compound, petruscine, on the kinetics of agelatin crosslinking reaction is described with regard to the belowillustrative, non-limiting examples. Petruscine slowed down thecrosslinking reaction in a dose dependent manner, suggesting that itserves as a substrate for transglutaminase and is crosslinked with thegelatin. It also increased the elasticity.

UHT Sterilization of Cross-Linkable Protein or Polypeptide

According to some embodiments of the present invention, ultra-hightemperature sterilization (UHT) processing is used to sterilize aprotein or polypeptide solution, such as a gelatin solution, inpreparation for its use in a medical application. UHT is thesterilization of a material in liquid form by heating it for a shorttime, from around 3-180 seconds, to a temperature range of 120-140° C.The high temperature reduces the processing time, thereby reducing thespoiling of material properties. Precise temperature control in the UHTsystem ensures requisite bioburden reduction in the material.

Successful, repeatable heat sterilization of a protein solution requiresthat the entire amount of solution is brought to a particulartemperature and held at that temperature for a set amount of time(acceptable time/temperature combinations are defined under GMPstandards). This is defined as “hold time.”

In an autoclave, the material typically is kept in the autoclave for30-40 minutes to ensure that all parts of the materials have achievedthe required temperature (generally about 121° C.) for at least thelength of the hold time.

In UHT, the process is far more controlled and thus can be much quicker.Rather than undergoing heating all at once, the material undergoes acontinuous flow process wherein small amounts of the material are beingheated at any given time.

According to some embodiments of the present invention, traditional UHTis used to partially or fully sterilize the protein solution componentof the herein described novel composition. In traditional UHT, theheating process for any particular aliquot of material takes a fewseconds and then that material is kept at the temperature for therequisite hold time.

According to another embodiment of the present invention, the proteinsolution undergoes a process of deaeration prior to entry into the UHTsystem.

According to a preferred embodiment of the present invention, heating ofthe material in the UHT system is accomplished by indirect heating ofthe material, for example through the heating of water or steam.

According to another embodiment of the present invention, heating of thematerial in the UHT system is accomplished by direct heating of thematerial, for example through the injection of heated steam.

According to a preferred embodiment of the present invention, when UHTprocessing is conducted with direct steam injection heating, the proteinor polypeptide solution being processed is initially prepared at aconcentration higher than the concentration required for the finalmedically-useful protein/polypeptide-crosslinker composition.

In a more preferred embodiment of the present invention, the initialconcentration of the protein/polypeptide solution being processed isabout 5% (w/v) higher than the desired final concentration of thatsolution.

According to some embodiments of the present invention, microwave UHT isused, wherein the heating process for each aliquot takes less than 1second, preferably less than 0.5 seconds, and more preferablyapproximately 2/10 second prior to the hold time. This short heatingtime is made possible by ensuring that every part of the material passesthrough a uniform field of heat waves at a constant flow rate. This is avery controlled and extremely accurate process.

Successful sterilization of a gelatin solution for use in the presentinvention requires that the heating process does not result in anyhydrolysis of the gelatin molecules. Autoclaving of protein solutions,for example gelatin solutions, is known to result in partial hydrolysisand a significant loss of cross-linking activity. Furthermore,autoclaving of gelatin solutions including materials such as urearesults in a near total loss of cross-linking activity of the gelatinmaterial. In other words, the extended heating period required forsuccessful autoclaving results in certain undesirable effects on thegelatin solution material.

For protein processing, UHT is many times more delicate than autoclavingsince the heating time required is far shorter than the time required inautoclaving. UHT does not result in significant partial hydrolysis ofgelatin molecules in gelatin solutions.

Direct heating of the protein or polypeptide solution during UHTprocessing is the more rapid form of heating in traditional UHT.However, direct heating requires the injection of steam directly intothe material. Normally, the steam is removed by vacuum once the materialhas been sterilized. However, this is impossible in a high viscosityprotein solution such as a gelatin solution. Therefore, if direct steaminjection heating is used, it is necessary to calibrate the UHT systemto assess by what percentage the material being sterilized is diluted bythe injected steam. Generally, this percentage is approximately 5%. Oncethe precise dilution percentage is determined, the initial solutions canbe made more concentrated that is required to allow for dilution bysteam injection.

Indirect heating, which takes about 20-30 seconds and is thus slightlyslower than direct heating, is simpler than direct heating as it doesnot at all affect the content of the material being processed.

Although uniform microwave field UHT is a very new technology, it isalso a very controlled and delicate sterilization method for use withprotein solutions. Microwave UHT is dependent on uniform microwave wavetechnology, a relatively new technology. This technology allows analiquot of material to be uniformly heated to a very specifictemperature. The entire amount of the material is heated to thatparticular temperature. None of the material gets any hotter than thattemperature and none escapes being heated to that temperature. Theentire UHT process is dependant on the reliability and consistency ofthis uniform heating method. A standard microwave is not capable ofcreating a uniform field of heat waves. The result is that heating isalways uneven to some degree. In a sterilization process that wouldensure that the entire material is sufficiently heated, some sections ofthe material would get overheated and be unsuitable for use.

Microwave UHT minimizes the overall process time by accomplishing therapid heating of direct steam injection heating without requiring anychanges in concentration to the material being processed.

Deaeration of a solution prior to UHT processing can be greatlyadvantageous since air bubbles in a material undergoing UHT processingwill burst over the course of the process. This can disrupt the processand result in uneven results of the sterilization heat process.

Preferentially, UHT processing of the protein or polypeptide solution isdone using a system designed for miniature thermal processing, definedas continuous flow processing with a flow rate of less than 2 L/min.Even more preferentially, the UHT process is carried out with a flowrate below 1.2 L/min. Flow rates of this magnitude are optionally andpreferably achieved using miniature traditional or uniform microwave UHTsystems, such as those supplied by Microthermics (Raleigh, N.C.). A lowflow rate allows for a more accurate and efficient UHT process.

Amine Donors

According to some embodiments of the present invention where primaryamine groups are a substrate for the crosslinker material, anamine-donor is added to the gelatin solution to modify the crosslinkingreaction kinetics or the mechanical properties of the crosslinkedprotein composition.

Polyamines are organic compounds having two or more primary aminogroups. Owing to their chemical nature, polyamines can form hydrogen,ionic, or covalent linkages with other molecules. In animals,post-translational covalent linkages of polyamines to numerous proteinshave been demonstrated, with much evidence indicating that thesereactions are catalyzed by transglutaminases that form cross-linkedcomplexes with two or one peptide-bound glutamine residues respectively(Serafinie-Fracassini D, et al. Plant Physiol. (1988) 87, 757-761.Ohtake Y, et al. Life Sciences 2007; 81, 7: p. 577-584).

Examples of suitable polyamines include but are not limited topoly-lysine, chitosan, or polyethylenimine.

Another suitable polyamine is polyvinylamine, a commercialamine-reactive PVA substance produced by BASF (Germany). The chainlength and charge density of polyvinylamine molecule can be varied toobtain different characteristics of a copolymer incorporatingpolyvinylamine.

In some embodiments, the polyamine is optionally included in the proteincomposition prior to mixture of the protein with the crosslinkermaterial.

In some embodiments, the polyamine is optionally included in thecrosslinker composition prior to mixture of the protein with thecrosslinker material.

In one embodiment, poly-lysine, a polymer of lysine that carriesmultiple positive charges and is used to mediate adhesion to livingcells, is included. The interaction with living cells is mediatedthrough negatively charged sialic acid carbohydrates found on membranesof most mammalian cells. Another, non-limiting example of a polyaminethat can be relevant for this purpose is polyethyleneimine.

In some embodiments, the polyamine is optionally added aftercrosslinking has begun. Normally, in type A gelatin there are moreglutamines than lysines, 48 vs. 30 residues, respectively, per each 1000amino acid residues of gelatin. Inclusion of polyamines in thecomposition can tilt the balance towards excess of primary amines. Theprimary amines can serve as anchor points for attachment into thetissue.

The addition of polyamines to the composition of the present inventioncan increase the cohesive strength of the composition as well as itsadhesiveness, by increasing the number of reactive sites in thecomposition.

In addition, polyamines may form flexible crosslinking bridges betweengelatin chains, thus increasing the flexibility of the crosslinked gel.

Also, polyamines have been shown to bind tissue fibronectin (see abovereferences). Thus, polyamines incorporated into a protein-crosslinkercomposition can also act as intermediate agents that connect thecomposition to native tissue.

In a preferred embodiment of the present invention, polyethylenimine(preferably branched polyethylenimine) is optionally included in theprotein composition.

Branched polyethyleneimine is a highly branched polymer with primaryamine groups, secondary amine groups, and tertiary amine groups.

Example 29 describes the use of polyethylenimine (PEI), for example andwithout limitation, to increase the elasticity of a mTG-crosslinkedgelatin composition.

Ammonia Scavenging, Sequestering and Binding Agents

Ammonia is highly toxic. Normally blood ammonium concentration is <50μmol/L, and an increase to only 100 μmol/L can lead to disturbance ofconsciousness. A blood ammonium concentration of 200 μmol/L isassociated with coma and convulsions.

Ammonium is produced in most cells of the body, as a result ofdeamination of amino acids and amines. The toxicity of ammonium abovethreshold concentrations of ammonium is due to the action of the enzymeglutamate dehydrogenase. This enzyme catalyses the oxidative deaminationof glutamate to ammonium and ketoglutarate; the reaction is readilyreversible, and the direction of reaction (towards deamination ofglutamate or glutamate formation) depends on the relative concentrationsof the various substrates. As the concentration of ammonium rises, sothe reaction proceeds in the direction of formation of glutamate fromketoglutarate. Ammonia intoxication occurs when blood ammonium risesbecause the capacity to detoxify it by formation of glutamate andglutamine has been exceeded.

Molecules of ammonia are released by the cross-linking reactionfacilitated by transglutaminases on fibrin, gelatin, and other proteins.Therefore, effecting a large amount of mTG cross-linking in a localphysiological environment could, in extreme situations, result in therelease of a toxic level of ammonia.

In an embodiment of the use of a gelatin-mTG composition in aphysiological context, such as an implantable and/or surgical context,the local levels of ammonia are preferably reduced below the potentiallydangerous threshold by the incorporation of an ammonia-scavenger agent,an ammonia-binding agents, or other ammonia-neutralizing agent in thegelatin-mTG composition. Such an agent could be included in either thegelatin component or mTG component that are mixed to form thegelatin-mTG composition.

A non-limiting example of such an agent is disaccharide lactulose.Lactulose is a synthetic disaccharide that is not hydrolysed byintestinal enzymes. Lactulose inhibits bacterial ammonia production byacidifying the content of the bowel. It promotes growth of colonicflora. The growing biomass uses ammonia and nitrogen from amino acids tosynthesise bacterial protein, which in turn inhibits protein degradationto NH₃. Lactulose leads to less ammonia by inhibiting bacterial ureadegradation and reduces colonic transit time, thus reducing the timeavailable for ammonia production and expediting ammonia elimination.(Deglin J H, et al. Lactulose. In Davis's drug guide for nurses (9thed., 2003) (pp. 589-590). Philadelphia: F. A. Davis.) Lactulose iscommercially available from Solvay SA (Brussels), among other suppliers.

Another embodiment of this invention optionally and preferably featuresa mixture of four forms of the strong cation exchange resin, Amberlite™IR-120 (Advanced Biosciences, Philadelphia, Pa.), in the treatment ofammonia intoxication. This resin mixture, with a total quantity of 750mEq, when used in the extracorporeal circulation system, was found to beefficient in the correction of hyperammonemia of experimental dogs andto be unaccompanied by any untoward effects. (Juggi J S, et al. In-VivoStudies with a Cation Exchange Resin Mixture in the Removal of ExcessiveAmmonium from the Extracorporeal Circulation System. ANZ J Surg 1968; 38(2): p 194-201).

Another embodiment of this invention optionally and preferably featuressaponins, particularly yucca saponin, or the glyco-fraction derivativeof Yucca shidigera plant, both of which have demonstratedammonia-binding ability (Hussain I, Ismail A M, Cheeke P R. Animal FeedScience and Technology, 1996; 62 (2), p. 121-129).

Another embodiment of this invention optionally and preferably featuresa sodium phenylacetate and sodium benzoate solution as an ammoniascavenger. Such a solution is commercially available in a non-limitingexample under the trade name AMMONUL® (Medicis, Scottsdale, Ariz.),which consists of a solution of 10% sodium phenylacetate, 10% sodiumbenzoate.

In another embodiment of the present invention, L-glutamine (L-Gln) orL-glutamate (L-Glu) is added to the protein-crosslinker composition,preferably to the protein component of the composition. L-Gln and L-Glustimulate the metabolism of ammonia to urea in cells, and also inhibitthe uptake and facilitates the extrusion of ammonia from cells (NakamuraE, Hagen S J. Am. J of Phys. GI and Liver Phys, 2002; 46(6), p.G1264-G1275). Without wishing to be limited by a single hypothesis, inan in situ cross-linking process that releases ammonia, L-Gln and/orL-Glu have utility in neutralizing the released ammonia by reducing theamount of free ammonia in the environment, thereby reducing the amountabsorbed by cells and accelerating the cells' natural ability tometabolize ammonia. The tissue response to a mTG-crosslinked gelatincomposition over its initial 14 days of subcutaneous implantation inrats was significantly improved by the inclusion of glutamate tosequester the Ammonia released by the mTG crosslinking reaction, asdescribed with regard to the below illustrative, non-limiting examples.

Coloring Agents

In another embodiment of the present invention, a biocompatible coloringagent is added to either the protein or cross-linker solution to improvethe visibility of the composition upon application.

Additional Hemostatic Agents

According to some embodiments of the present invention, any of thecompositions described above may further comprise an additionalhemostatic agent, which may be selected from the group consisting ofcoagulation factors, coagulation initiators, platelet activators,vasoconstrictors, and fibrinolysis inhibitors. Examples of these includebut are not limited to epinephrine, adrenochrome, collagens, thrombin,fibrin, fibrinogen, oxidized cellulose, and chitosan.

Configurations of Lyophilized Product

In an embodiment of the present invention, a dried or frozen compositionis formed wherein the cross-linkable protein or polypeptide isthoroughly mixed with the non-toxic cross-linker to form a homogenoussolution and the temperature of the solutions is reduced immediately toprevent completion of the cross-linking process. The mixed compositionis then either frozen or frozen and dried to form a novel, uniformcomposition.

This type of composition has great utility in that it allows for theprecise control of the time that it takes for the composition to for acohesive gel in situ. As can be seen in the viscometer graphs presentedin the examples, the time that it takes for the cross-linking to occurcan be very precisely defined. Since the activity of some cross-linkers,such as mTG, is temperature dependant, if the temperature of thecomposition is reduced below the active temperature of the cross-linker,the cross-linking process can be effectively halted. In the case of mTG,cross-linking activity is essentially halted below a temperature ofapproximately 20° C.

In a preferred embodiment of this invention, the reaction is stoppedbefore the composition achieves 30% of its ultimate mechanical strength.

In a more preferred embodiment of this invention, the reaction isstopped before the composition achieves 15% of its ultimate mechanicalstrength.

In an even more preferred embodiment of this invention, the reaction isstopped before the composition achieves 5% of its ultimate mechanicalstrength.

Ultimate mechanical strength, for the sake of this embodiment, isdefined as the point at which the all of the composition'scross-linkable material has been cross-linked to a sufficient degree soas to not be freely flowable. In viscometer testing, this point occursroughly at 10M cP.

In an embodiment of the present invention, a dried composition is formedwherein the dry crosslinker material is thoroughly dispersed through alyophilized composition of cross-linkable protein or polypeptide.

In a preferred embodiment, the protein is gelatin and a non-crosslinkedgelatin foam is lyophilized prior to dispersal of crosslinker throughoutsuch a porous foam.

In another preferred embodiment, dry crosslinker material is added tothe gelatin foam such that the crosslinker does not dissolve into thefoam (ie no crosslinking activity is observed prior to lyophilization).

It was surprisingly found that a reconstitutable foam could optionallybe formed from a gelatin solution that was sufficiently stabile so as toallow for the lyophilization of the gelatin in foam form without theaddition of any stabilizing or crosslinking agents.

In a preferred, illustrative embodiment of the present invention forforming such a foam, a gelatin solution is prepared and held at atemperature where it is in liquid form. The gelatin solution is thensubjected to an extended and preferably continuous foaming process whileit is cooled to a temperature below its sol-gel transition point.

The concentration of gelatin solution is preferably in the range of0.5%-20% w/w, more preferably 5-10% w/w.

The initial temperature of the gelatin solution is 30° C.-70° C.,preferably 30° C.-50° C., and more preferably 35° C.-40° C. Theenvironmental temperature during the foaming process is 0° C.-25° C.,preferably 15° C.-25° C. and more preferably 20° C.-23° C. Non-limitingexamples of foaming processes include stirring, mixing, blending, andinjection of a gas.

Preferably, the foaming process includes stirring or mixing.

One or more foaming techniques may optionally be used in the foamingprocess. Alternatively, one foaming technique may optionally be usedmultiple times under different conditions: for example, gentle stirringto generate a low level of foam following by vigorous stirring toachieve maximal aeration in the gelatin foam.

In an optional embodiment, upon the completion of foaming, the gelatinfoam is preferably transferred to a vessel that had been cooled to atemperature lower than the temperature of the gelatin foam uponcompletion of the foaming process.

In another embodiment, the gelatin foam is optionally and preferablyrapidly cooled immediately upon completion of foaming process. Anon-limiting example of rapid cooling is exposing gelatin foam to liquidnitrogen immediately after the foaming process.

In a preferred embodiment, the dry gelatin foam contains less than about12% moisture. In a more preferred embodiment, the dry gelatin foamcontains less than about 8% moisture.

In another embodiment, the gelatin foam is optionally not furtherstabilized by cooling or other method immediately (within up to about 5minutes) upon the completion of foaming such that the foam partiallycollapses resulting in the formation of a denser layer of gelatin foamon the bottom of the foam.

In an embodiment of the above, the denser layer optionally comprisesless than about 50% of the thickness of the lyophilized gelatincomposition, preferably less than about 35%, and more preferably lessthan about 20%.

Density as used here refers to an increase in the weight of gelatin pervolume of lyophilized composition. Such an increase can optionally be aslittle as 5% but is preferably greater than about 10% and morepreferably greater than about 20%.

Without wishing to be limited to a single hypothesis or to a closedlist, it is believed that such a dense layer of gelatin foam providesmechanical strength to the lyophilized gelatin composition withoutaffecting the reconstitution profile of the top part of the drycomposition.

EXAMPLES

Reference is now made to the following examples, which together with theabove description, illustrate some embodiments of the invention in a nonlimiting fashion.

Example 1 Comparison of Cross-Linking Time Using Acetate Buffer andCitrate Buffer Materials

The following materials were used in the experiment: 300 bloom, type Aporcine gelatin (Sigma, St. Louis, Mo.), Gelatin Medex—300 bloom 70mesh, pharmaceutical gelatin [Medex, England batch], 98% urea [AlfaAesar, Lancester], Calcium Chloride 97% dried powder [Alfa Aesar,Lancester], 0.1M Sodium Acetate buffer (pH 6.1), 0.5M Sodium Citratedehydrate 99% [Alfa Aesar, Lancaster], D-Sorbitol 97% [Sigma, St. Louis,Mo.], 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme, 90%maltodextrin) [Ajinomoto, Japan].

Methods

Stock solutions of 2M Calcium solution, 4.5M and 5M urea, 0.1 M SodiumAcetate solution pH 6.0, 0.5M Sodium Acetate solution pH 6.0, 2M SodiumCitrate solution pH 6.0 and, 2.36M Citric Acid solution was prepared.

25% (w/w) Gelatin solution with 2M urea, 1M Calcium, 0.1M Sodium Acetate(solution A) was prepared. 7.5% (w/w) microbial transglutaminase (10%w/w mTG—ACTIVA-TG) solutions were prepared by dissolving mTG in thefollowing different solutions:

Solution 1—0.5M Sodium Acetate Solution 2—0.5M Sodium Citrate ViscometerTests

For each viscometry test, 25 mL of gelatin solution was mixed with 12.5mL of mTG solution in a 50 mL beaker. The viscosity of the mixedgelatin-mTG solution was then tracked as it underwent gelation.Different test groups were compared by recording the time required foreach test group to achieve 30% and 90% of the maximum viscosity able tobe recorded by the viscometer at the specific speed and with thespecific spindle used for that test.

In this experiment, a DV II+ PRO Digital Viscometer (BrookfieldEngineering, Middleboro, Mass.) was used with a T-E 95 “t-bar” spindle.A helipath viscometer stand was used to maintain vertical movement ofthe spindle over the course of the viscometer test. The helipath movedalong a 1 cm path. The viscometer readings were outputted by theviscometer and read using HyperTerminal software at a rate of 1 readingper second. The rotational speed of the spindle for the viscometry testwas 0.5 rpm. The maximum recordable viscosity at this speed with the T-E95 spindle was 10×10⁶ cP, meaning that the 30% point was equivalent to3×10⁶ cP and the 90% point was equivalent to 9×10⁶ cP.

The beaker was submerged in a 37° C. water bath for the entire extent ofthe viscometer test. Average temperature within the beaker also recordedthroughout the test to ensure consistency between test groups.

Results

FIG. 2 and Table 1 show viscosity changes of 25% (w/w) gelatin in 1MCalcium 2M urea (solution A) reacted with 7.5% (w/w) ACTIVA-TG 10% in0.5M Sodium Acetate (solution 1) and 0.5M Sodium Citrate (solution 2).

TABLE 1 Comparison of viscosity changes with time of mTG buffers Timefor 30% of Time for 90% of Solution maximal viscosity, sec maximalviscosity, sec Solution A reacted with 127 168 solution 1 Solution Areacted with 187 234 solution 2

The results of this experiment demonstrated that use of mTG in 0.5MSodium Acetate led to a significantly shorter cross-linking timecompared to mTG in 0.5M Sodium Citrate.

Example 2 Effects of Different Buffers and Buffer Ion Concentrations onCross-Linking Time Materials

The following materials were used in the experiments: 300 bloom, type Apharmaceutical porcine gelatin (70 mesh) [Ital Gelatine, Santa Vittoriad'Alba, Italy], Urea—minimum 99.5%, [Sigma, St. Louis], Calcium chloride97% dried powder [Alfa Aesar, Lancester], Sodium Citrate Dehydrate 99%[Alfa Aesar, Lancaster], citric acid anhydrous [Frutarom, Israel],Sodium Acetate Trihydrate [Sigma, St. Louis], Acetic Acid Glacialanalytical grade [Frutarom, Israel] and, 10% microbialTransglutaminase—ACTIVA-TG 10% (10% enzyme, 90% maltodextrin)[Ajinomoto, Japan].

Method

The following stock solutions were prepared: 2M of Calcium solution, 4Mand 4.5M urea solutions, 0.1M sodium acetate solution, 2M of Sodiumcitrate solution and 2.36M Citric acid solution.

A 25% (w/w) gelatin solution in 2M urea and 1M CaCl₂ (solution A) wasprepared. 7.5% (w/w) microbial transglutaminase was prepared anddissolved into the following six solutions:

Solution 1—0.1 M Sodium Acetate Solution 2—0.25 M Sodium AcetateSolution 3—0.5 M Sodium Acetate Solution 4—0.25 M Sodium CitrateSolution 5—0.5 M Sodium Citrate Solution 6—0.6 M Sodium Citrate

To determine cross-linking (XL) time, 2 mL of gelatin solution was mixedwith 1 mL of each type of mTG solution. For each type of mTG solution, 3separate samples were prepared in separate wells of a 12-well cultureplate. Solutions were thoroughly mixed to form a homogenous solution andthen cross-linking time was determined by time at which mixed solutionformed a coherent, gelatinous mass.

Results

As shown in Table 2, it was found that lower ion concentrations (0.1Minstead of 0.5M) in the mTG buffer results in quicker cross-linking bothwith Sodium Acetate and with Sodium Citrate buffer.

TABLE 2 Cross linking of gelatin using mTG solutions in different ionicstrengths results. XL refers to cross-linking. Test No 1 Test No 2Enzyme solution Time to XL Description Time to XL Description Summary 12 min XL begins after 2 min XL begins A 0.1M Sodium 1 min after 1 minhomogenous Acetate gel is formed after 2 min. 2 2.5 min   After 1.5 min2.5 min   After 2.5 min A 0.25M Sodium XL begins. XL begins. homogenousAcetate gel is formed after 2.5 min. The gel becomes brittle with time.3 4 min After 1 min XL 4 min After 1 min A 0.5M Sodium begins. The XLbegins. homogenous Acetate formed gel is The formed gel is formed verygel is very after 4 min. homogenous. homogenous. However. the gelbecomes brittle after short period of time. 4 2.5 min   Starts to XL 2.5min   Starts to XL A gelatinous 0.25M Sodium after 1.5 min. after 1.5min mass is Citrate formed after 2.5 min. 5 4 min Starts to XL 4 minStarts to XL Forms a 0.5M Sodium after 1.5 min. after 1.5 min.gelatinous Citrate mass after 4 min. 6 4 min Non uniform 4 min Nonuniform Starts to gel 0.6M Sodium gelation-part gelation-part after 2min. Citrate gelled gelled After 4 min a immediately immediately firm,flexible and the rest and the rest gel is formed. started to gel startedto gel after 2 min. after 2 min.

The results in Table 2 show a direct correlation between the ionicstrength and cross-linking time, wherein the lower ionic strengths inthe mTG solution buffer resulted in more rapid cross-linking. mTGsolutions in sodium acetate buffer cross-linked more rapidly and formedmore homogenous gels than those in sodium citrate buffer. Enzymesolutions with sodium citrate buffer cross linked more slowly and formeda non-homogenous gel, probably as a result of the physical gelatingelation caused by the sodium citrate. However, the gels formed with mTGin sodium citrate are much more flexible and cohesive compared to gelswith sodium acetate.

Example 3 Effect of Ionic Strength of Gelatin Buffer Solution onCross-Linking Time Materials

The following materials were used in the experiments: 300 bloom type Aporcine gelatin [Sigma, St. Louis, Mo.], Sodium Acetate—0.1M SodiumAcetate buffer (pH 6.0), Sodium Acetate—0.25M Sodium Acetate buffer (pH6.0), Sodium Acetate—0.5M Sodium Acetate buffer (pH 6.0), [SigmaAldrich] and, 10% Microbial transglutaminase—ACTIVA-TG 10% (10% enzyme,90% maltodextrin) [Ajinomoto, Japan].

Method

25% (w/w) gelatin in 0.1M sodium acetate buffer (solution A), 25% (w/w)gelatin in 0.25M sodium acetate buffer (solution B), 25% (w/w) gelatinin 0.5M sodium acetate buffer (solution C), 7.5% (w/w) ACTIVA-TG in 0.1MNa—Ac buffer (solution 1 and 7.5% (w/w) ACTIVA-TG 0.25M Na—Ac buffer(solution 2) were prepared.

Results

It was found that raising ion concentration to 0.25-0.5M of gelatinbuffer decrease cross-linking time. However, gels did not form when bothgelatin buffer and mTG buffer are of high ion concentration.Furthermore, cross-linked gels formed with gelatin solutions in high ionconcentration buffer were found to be less thermally stable than gelsformed at low ion concentrations.

As illustrated in Tables 3, 4, and 5, increasing ionic strength ofgelatin solutions with mTG solutions of 0.1M, decreased cross-linkingtime hence improving reaction time. No notable differences were observedin formed gels of 0.25M and 0.5M as compared to formed gel of 0.1MNa—Ac.

Increased ionic strength of mTG solutions with increased ionic strengthof gelatin solutions provided mechanically weaker cross-linked gels withlonger gel forming reaction times.

TABLE 3 Transition temperature examination Temperature Physical state ofPhysical state of Physical state of range, ° C. solution A solution Bsolution C 45-46 liquid liquid liquid 40-41 liquid liquid liquid 38-39liquid liquid liquid 36-37 liquid liquid liquid 34-35 liquid liquidliquid 32-33 Medium viscosity Medium viscosity Medium viscosity 31-32High viscosity High viscosity Very high viscosity/ solid

TABLE 4 Cross-linking examination - summary of experimental results withmTG solution 1 (0.1M Na—Ac). Gelatin Gelation Time solution (min)Description of Cross-Linked Gel A 0:35 Minor adhesion and elasticity arenoticed in the 0.1M formed gel. Gel is brittle. sodium After 10 minutesgel is not adhesive or elastic. acetate Brittles fast. B 0:20 Gel hadgood adhesion property, but lacks 0.25M elasticity. After 10 minutes gelremains with sodium good adhesion properties but becomes acetate verybrittle. C 0:15 Formed gel resembles properties of solution B. 0.5MAfter 10 minutes formed gel loses adhesion, sodium becomes very brittleand is not elastic. acetate

TABLE 5 Summary of experimental results with mTG solution 2 (0.25MNa—Ac). Gelatin Gelation Time solution (min) Description of Cross-LinkedGel A 0:25 Formed gel is soft, with no adhesion properties. 0.1M Veryweak and brittle. sodium After 10 minutes, gel becomes very brittle andacetate does not improve in any mechanical property. B N/A Gel issemi-liquid and not fully formed for a 0.25M long period of time (about2 minutes). Very sodium soft and very weak, and does not improve acetatesignificantly with time. C N/A Similar to solution B. Gel appears to benot 0.5M fully formed for long period of time sodium (about 2 minutes)and only about half of it acetate cross-linked to solid state. Time doesnot change significantly mechanical properties of the gel.

Example 4 Effect of Calcium Chloride and Urea on Transition PointMaterials

The following materials were used in the experiments: 300 bloom type Aporcine gelatin [Sigma, St. Louis, Mo.], 98% urea [Alfa Aesar,Lancester], Calcium Chloride 97% [Alfa Aesar, Lancester], PBS—Dulbecco'sPhosphate Buffered Saline without Calcium and Magnesium [BiologicalIndustries, Israel], Microbial Transglutaminase ACTIVA-WM, 1% enzymepowder in maltodextrin [Ajinomoto, Japan], 10% microbialTransglutaminase—ACTIVA-TG 10% (10% enzyme, 90% maltodextrin)[Ajinomoto, Japan].

Methods

Stock solutions of 5 M Calcium solution, 5 M of Urea solution wereprepared. 25% (w/w) gelatin solution, urea and calcium solutions wereprepared by diluting urea and calcium stock solutions.

Control A—gelatin was dissolved in PBSControl Calcium A—gelatin was dissolved in 2 M Calcium.Control Calcium B—gelatin was dissolved in 1 M calcium.Solution C—gelatin was dissolved in PBS solution containing 1 M calciumand 2 M urea.Solution D—gelatin was dissolved in PBS solution containing 1 M calciumand 3 M urea.Solution E—gelatin was dissolved in PBS solution containing 0.5M calciumand 2M urea.Solution F—gelatin was dissolved in PBS solution containing 0.5 Mcalcium and 3 M urea.

Results

As shown in Table 6, urea and calcium had a synergistic effect onreducing the transition point of 25% (w/w) gelatin solutions. 25% (w/w)gelatin solution containing 1 M Calcium chloride combined with 2 M ofUrea has a low viscosity at RT. 25% (w/w) gelatin solution containing0.5 M Calcium chloride and 3 M urea is viscous at RT. Cross linking ofgelatin gels containing urea provided weaker gels. The higher the ureaconcentration, the weaker the gel that was formed. The presence ofcalcium in the gelatin solutions increased the cross linked gelstrength.

TABLE 6 Summary of sol-gel transition results for gelatin gels at 24° C.Gelatin Solution Additives State at 24° C. Description Control — GelledClear gel Control 2M Ca Liquid Opaque solution Calcium A Control 1M CaHighly viscous Opaque gel Calcium B Solution C 1M Ca 2M urea LiquidOpaque solution Solution D 1M Ca 3M Urea Liquid Opaque solution SolutionE 0.5M Ca 2M urea Highly viscous Opaque solution Solution F 0.5M Ca 3MUrea Slightly Viscous Opaque solution

Example 5 Optimization Experiments Done to Determine the AppropriateAmount of Cross-Linker to Use for Each Combination of Gelatin and mTGSolutions Materials

The following materials were used in the experiment: Gelita 300 bloom,type A porcine gelatin (Medex, England), Urea 99.5% (Sigma-Aldrich, St.Louis), Calcium Chloride (Alfa Aesar, Lancester), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),ACTIVA TG (10% protein, 90% maltodextrin) microbial transglutaminase(Ajinomoto, Japan), Sodium Citrate (Sigma-Aldrich, St. Louis), CitricAcid Monohydrate (Sigma-Aldrich, St. Louis), D-Sorbitol, 97% (SigmaAldrich St. Louis, Mo.).

Stock Solution Preparation:

2M of Calcium solution was prepared in 0.1 M Na—Ac pH 6.0. The solutionwas filtrated using number 1 Whatman filter paper.

4M and 5 M urea solutions were prepared in 0.1 M Na—Ac pH 6.0. Thesolutions were filtered using 250 mL filter system with 22 um celluloseacetate membrane and kept refrigerated.

0.1 M Na—Ac solution pH 6.0 was prepared and filtered using 250 mLfilter system with 22 um cellulose acetate membrane and keptrefrigerated.

2M of Sodium citrate solution was prepared.

2.36M Citric acid solution was prepared

Gelatin Solution Preparation:

25% (w/w) gelatin solutions were prepared in different additives asfollows:

Solution A—in 2 M urea, 1 M CaCl₂, 0.1 M Na—Ac pH 6.0.Solution B—in 4.5 M urea, 0.1 M Na—Ac pH 6.0.

In order to completely dissolve the gelatin powder, the solutions wereheated to 50° C. and vigorously stirred. The solution was then passed to24° C. incubator and kept there overnight (ON) until use.

Microbial Transglutaminase Solutions Preparation:

Microbial transglutaminase (mTG) solutions were prepared, by dissolvingmTG (ACTIVA-TG 10%) in different concentrations, in different solutions.The solutions were prepared immediately before use. In order tocompletely dissolve mTG, the solution had to be vigorously stirred usinga plastic rod. The solutions were prepared as follows:

Solution 1—7.5% (w/w) of ACTIVA-TG 10% in 0.1 M Na—AcSolution 2—5% (w/w) of ACTIVA-TG 10% in 0.1 M Na-ASolution 3—6% (w/w) of ACTIVA-TG 10% in 0.1 M Na-ASolution 4—7% (w/w) of ACTIVA-TG 10% in 0.1 M Na—AcSolution 5—5% (w/w) of ACTIVA-TG 10% in 3:1 sorbitol (ratio of drysorbitol weight to dry gelatin weight) with 0.1M Na—AcSolution 6—6.25% (w/w) of ACTIVA-TG 10% in 3:1 sorbitol with 0.1 M Na—AcSolution 7—7.5% (w/w) of ACTIVA-TG 10% in 3:1 sorbitol with 0.1M Na—AcSolution 8—10% (w/w) of ACTIVA-TG 10% in 0.1 M Na-CitrateSolution 9—12.5% (w/w) of ACTIVA-TG 10% in 0.1 M Na-CitrateSolution 10—2.5% (w/w) of ACTIVA-TG 10% in 0.1 M Na-CitrateSolution 11—5% (w/w) of ACTIVA-TG 10% in 0.1 M Na-CitrateSolution 12—7.5% (w/w) of ACTIVA-TG 10% in 0.1 M Na-CitrateSolution 13—3% (w/w) of ACTIVA-TG 10% in 0.1 M Na-CitrateSolution 14—5% (w/w) of ACTIVA-TG 10% in 3:1 sorbitol (sorbitol togelatin ratio) with 0.5M Na-CitrateSolution 15—6.25% (w/w) of ACTIVA-TG 10% in 3:1 sorbitol (sorbitol togelatin ratio) with 0.5M Na-Citrate

Viscometer Testing:

Viscometer experiments were conducted according to the proceduredescribed above.

Results

Solutions were examined via viscometer and the optimal enzymeconcentration for each gelatin-mTG solution was determined. For eachsolution, the average temperature throughout the experiment, the time to30% of the torque and time to 90% of the torque were examined.

FIG. 3 displays the time to viscosities of 3×10⁶ cP (30% of fully formedgel) and 9×10⁶ cP (90% of fully formed gel) for the differentformulations mentioned above.

FIG. 3A shows the time to viscosities for the above solutions of 25%gelatin, with 4.5 M urea in 0.1 M Na—Ac. The first two bars relate tothe time to 30% and 90% of the torque for a concentration of mTG at 5%;the next two bars relate to the time to 30% and 90% of the torque for aconcentration of mTG at 6%; while the last two bars relate to the timeto 30% and 90% of the torque for a concentration of mTG at 7%, allrespectively. As shown, the time to both 30% and 90% of the torquedecreased with increasing concentrations of mTG, showing that increasedamounts of mTG increase the rapidity of cross-linking.

FIG. 3B shows the time to viscosities for the above solutions of 25%gelatin, with 4.5 M urea in 0.1 M Na—Ac, and sorbitol present in a 3:1ratio of sorbitol:gelatin. The first two bars relate to the time to 30%and 90% of the torque, respectively, for a concentration of mTG at 6.3%,while the last two bars relate to the time to 30% and 90% of the torque,for respectively, for a concentration of mTG at 7.5. Again, the time toboth 30% and 90% of the torque decreased with increasing concentrationsof mTG.

FIG. 3C shows the time to viscosities for the above solutions of 25%gelatin, with 2 M urea and 1 M calcium, in 0.1 M Na—Ac, with a singleconcentration of mTG at 7.5%. The left bar shows the time to 30% oftorque while the right bar shows the time to 90% of torque. Effectivecross-linking is also found under these conditions.

FIG. 3D shows the time to viscosities for the above solutions of 25%gelatin, with 2 M urea and 1 M calcium, in 0.1 M Na—Ac, and sorbitolpresent in a 3:1 ratio of sorbitol:gelatin. The first two bars relate tothe time to 30% and 90% of the torque, respectively, for a concentrationof mTG at 5%, the next two bars relate to the time to 30% and 90% of thetorque, for respectively, for a concentration of mTG at 6.3% and thelast two bars relate to the time to 30% and 90% of the torque, forrespectively, for a concentration of mTG at 7.5%. As compared to FIG.3C, sorbitol has clearly resulted in a greater rapidity ofcross-linking, as the time to torque has significantly decreased,although it is still at least somewhat dependent upon the concentrationof mTG.

FIG. 3E shows the time to viscosities for the above solutions of 25%gelatin, with 4.5 M urea, where the gelatin solution is in 0.1 M sodiumacetate buffer and the mTG is in 0.5 M sodium citrate buffer. The firsttwo bars relate to the time to 30% and 90% of the torque, respectively,for a concentration of mTG at 7.5%, the next two bars relate to the timeto 30% and 90% of the torque, respectively, for a concentration of mTGat 5% and the last two bars relate to the time to 30% and 90% of thetorque, for respectively, for a concentration of mTG at 2.5%. Increasedurea, without calcium, still results in a greater rapidity ofcross-linking, as the time to torque has significantly decreased,although it is still at least somewhat dependent upon the concentrationof mTG.

FIG. 3F shows the time to viscosities for the above solutions of 25%gelatin, with 4.5 M urea, in 0.1 M Na—Ac with 0.5 M sodium citrate, withthe addition of sorbitol in a 3:1 ratio with gelatin. The two barsrelate to the time to 30% and 90% of the torque, respectively, for aconcentration of mTG at 5%. Sorbitol appears to decrease the rate ofcross-linking and hence the time to torque, whether for 30% or 90%.

Example 6 Effect of Calcium Hydroxide on Protein Solution TransitionPoint

This Example shows the effect of calcium hydroxide on the sol-geltransition point of gelatin solution. Calcium hydroxide was shown toreduce this transition point.

Materials

The following materials were used in the experiment: 300 bloom, type Aporcine gelatin (Gelita, Sioux City), Calcium hydroxide (Sigma-Aldrich,St. Louis, Urea 98% (Alfa Aesar, Lancester), Sodium Acetate trihydrate(Sigma-Aldrich, St. Louis), Acetic Acid (Frutaron, Israel).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 4 M Urea and 2MCalcium hydroxide were prepared.

The following solutions were prepared:

Control—25% (w/w) Gelatin solution in 0.1M Sodium Acetate

Solution A—25% (w/w) Gelatin in 2M CaOH

Solution B—25% (w/w) Gelatin solution in 2M urea 1M CaOH

Solution C—25% (w/w) Gelatin solution in 0.5M CaOH

All solutions were heated to 50° C. while constant stirring was appliedto ensure formation of homogenous solution. After homogenous solutionwas achieved, all solutions were moved to 22° C. environment. After 2hours, solutions were manually palpated to assess physical state (liquidor gel).

Results

Solutions A and B were in liquid form at 22° C. while the controlsolution and solution C were in gel form, indicating that calciumhydroxide can decrease the sol-gel transition point of gelatin solutionand that this effect is dose dependant.

Example 7 Effect of Calcium Sequestering Agents on mTG Crosslinking ofGelatin Solutions

The effect on mTG-facilitated cross-linking of gelatin solutions whereEDTA or sodium citrate has been added to mTG solutions is described inthe below example.

Materials

The following materials were used in the experiments: 300 bloom type Aporcine gelatin [Sigma, St. Louis, Mo.], 98% urea [Alfa Aesar,Lancester], Calcium Chloride 97% [Alfa Aesar, Lancester], PBS—Dulbecco'sPhosphate Buffered Saline without Calcium and Magnesium [BiologicalIndustries, Israel], Ethylenediaminetetraacetic acid, Sodium Citratedehydrate 99% [Alfa Aesar, Lancaster], Citric acid anhydrous [Frutarom,Israel] Microbial Transglutaminase ACTIVA-WM, 1% enzyme powder inmaltodextrin [Ajinomoto, Japan], 10% microbialTransglutaminase—ACTIVA-TG 10% (10% enzyme, 90% maltodextrin)[Ajinomoto, Japan].

Methods

Stock solutions of 5 M Calcium solution, 5 M of Urea solution and, 2 Mof EDTA were prepared. 25% (w/w) gelatin solution, urea and calciumsolutions were prepared by diluting urea and calcium stock solutions.

Control A—gelatin was dissolved in PBSControl Calcium A—gelatin was dissolved in 2 M Calcium.Control Calcium B—gelatin was dissolved in 1 M calcium.Solution C—gelatin was dissolved in PBS solution containing 1 M calciumand 2 M urea.Solution D—gelatin was dissolved in PBS solution containing 1 M calciumand 3 M urea.Solution E—gelatin was dissolved in PBS solution containing 0.5 Mcalcium and 2 MSolution F—gelatin was dissolved in PBS solution containing 0.5 Mcalcium and 3 M urea.Solution G—gelatin was dissolved in PBS solution containing 4 M urea.0.2% w/w mTG solutions (20% w/w solutions of ACTIVA-WM: 1% mTG, 99%maltodextrin) were prepared as follows:mTG Control—mTG was dissolved in PBS.Solution 1—mTG was dissolved in 2 M EDTA solution in PBS. (Note: whiledissolving mTG in EDTA, an opaque solution, slightly white colored, wasformed. The mTG formed clumps that later dissolved in the solution)Solution 2—mTG was dissolved in 1.5 M of EDTA solution in PBS.Solution 3—mTG was dissolved in 0.75 M of EDTA solution in PBS.Solution 4—mTG was dissolved in a solution containing 2 M sodiumcitrate.Solution 5—mTG was dissolved in a solution containing 1 M sodiumcitrate.Solution 6—mTG was dissolved in a solution containing 0.5 M of sodiumcitrate.1% w/w concentrated mTG solutions (10% w/w solution of ACTIVA-TG: 10%mTG, 90% maltodextrin) solutions were prepared:Control—Concentrated mTG dissolved in PBS.Solution 7—Concentrated mTG dissolved in 0.5 M sodium citrate solution.

Results

Tables 7 and 8 below summarize the experimental results for gelatin gelscross linked (CL) with mTG. Table 7 describes gelatin gels cross linkedwith mTG containing EDTA and Table 8 describes gelatin gels cross linkedwith mTG containing sodium citrate. The effect of sodium citrate ongelatin solutions is summarized in Table 9.

TABLE 7 Cross linking of gelatin solutions using mTG containing EDTACross linking with mTG Solution 2 Solution 3 mTG Description- Solution 10.2% mTG 0.2% mTG Control 2M EDTA Gelatin 25% w/w 0.2% mTG in in 1.5M in0.75M (without without Solution gelatin plus: 2M EDTA EDTA EDTA EDTA)mTG Control — CL Was not CL CL after CL immediately, examinedimmediately ~3 min. immediately. formed a formed a Formed a Formed aclear, very clear, strong clear gel, clear, very strong and gel, verystrong, but strong and elastic gel, but elastic but not as elastic gelnot adhesive. not strong and but not adhesive. flexible as adhesive atwith all. EDTA. Very sticky. Control 2M Ca CL Was not Was not Weak gelWas not Calcium A immediately. examined examined was examined Formed aformed white, foamy after ~6 min. gel that Sticky. swelled. A bitsticky. Control 1M Ca CL Was not Immediately When Was not Calcium Bimmediately. examined partial cross examined examined Very foamy,linking was after 10 min white, non- observed. CL uniform gel. Stronggel. was not Strong yet not observed. adhesive. Solution C 1M Ca 2M CLCL after CL after 5 min. After 8.5 min Immediately urea immediately. 20sec. Created an weak, CL. White. Formed white, Non- opaque gel, opaquefoamy, foamy, uniform, not so gel was strong and swollen gel. white,strong as formed. flexible gel Was not swollen other gels Became isformed. sticky. Very gel was with EDTA. more The gel is strong & formed.adhesive not flexible. and strong adhesive. with time. Solution D 1M Ca3M CL CL A non- After 9.5 min Was not Urea immediately. immediate,uniform gel formed a examined. Formed a formed a is formed very white,swollen white, immediately. weak, gel. Not so foamy gel. This gel isopaque strong gel. Not sticky. not foamy. gel. CL completely after >7min. Solution E 0.5M Ca CL Foams After ~8 min CL after 4 min. Was not 2Murea immediately. immediately. a weak Formed a examined Formed a Startsto gel was weak gel. white, fluffy CL only formed, gel. Less after 3min. A opaque but foamy than weak gel not foamy. gels formed was with 1Mformed. EDTA and weaker. Solution F 0.5M Ca CL Immediately. A non CLafter Was not 3M Urea immediately. CL. Not uniform gel 3.5 min. examinedWhite, as foamy is formed Formed an swollen gel is or white as after 6min. opaque formed. Not with 2M Very weak. sticky gel. as foamy or EDTSNot as The gel is strong as with foamy as weak. 1M Ca. with 1.5M EDTA.

TABLE 8 Cross linking of gelatin solutions using mTG containing Sodiumcitrate Cross linking with mTG Description- Solution 4 Solution 5 25%w/w 0.2% mTG in 2M 0.2% mTG in 1M 2M Sodium Citrate Solution gelatinplus: Sodium citrate Sodium citrate without mTG Control 25% gelatin CLimmediately. After ~30 sec. a CL immediately, Formed a strongnon-uniform gel is forming a strong, and flexible gel formed. The gel isflexible gel. Not that in not not so strong or sticky. When heatedadhesive. After sticky gel. When to 37° C. - did not heating above 40°C. heated to 37° C. - did reverse. partially reversed. not reverse.Control 2M Ca Was not examined CL immediately, Was not examined CalciumA forming a white fluffy gel. Control 1M Ca Was not examined Was notexamined Was not examined Calcium B Solution C 1M Ca 2M Immediately Wasnot examined Immediately CL. urea formed a white, White, foamy, strongswollen gel. Very and flexible gel is strong and flexible. formed. Thegel is Not adhesive. A bit not adhesive. non-uniform and more brittlethan without the enzyme. Solution D 1M Ca 3M Was not examined Was notexamined Was not examined. Urea Solution E 0.5M Ca 2M Was not examinedWas not examined Was not examined urea Solution F 0.5M Ca 3M Was notexamined Was not examined Was not examined Urea

TABLE 9 Cross linking of gelatin solutions using mTG solutionscontaining sodium citrate Control A Solution C Solution G (25% (w/w)((25% (w/w) (25% (w/w) gelatin solution gelatin in 2M urea, gelatin in4M mTG solution in PBS) 1M Ca and PBS) urea and PBS) Conc. mTG control(1% CL within 1 min. CL within 2 min. CL within 2 min. (w/w) conc. mTGsolution in Formed a Formed a very formed a very PBS) sticky gel. Gelslimy, sticky, weak soft, flexible is quite brittle. and soft gel thatis weak gel. After very brittle. 13 min the gel appears to be strongerand still flexible, yet brittle. Conc. mTG Solution 7 Immediate CL.Immediately CL after 1.5 min. (1% (w/w) conc. mTG Very brittle gelformed a very Formed a very solution in 0.5M sodium is formed. flexible,sticky gel brittle and weak. citrate) after 1.5 mi. After Yet stickygel. 10 min the gel is very strong, flexible and sticky. With timebecomes brittle mTG solution 6 (0.2% CL after 30 sec. CL within 10 min.Was not (w/w) mTG solution in 0.5M Formed a very Formed a very examined.sodium citrate) strong and weak, non-uniform, flexible gel, yet flexiblegel. not sticky at all.

As shown in the Tables above, EDTA physically cross linked gelatinsolutions. High concentrations of EDTA (0.75-2 M) immediately crosslinked gelatin solutions. Gelatin gels cross linked with EDTA were notadhesive. EDTA formed very strong and flexible gels when mixed withgelatin. The higher the EDTA concentration, the stronger the gel thatwas formed. At moderate EDTA concentrations (0.5-0.75 M), non-homogenouscross linking occurred. This was probably due to insufficient blendingof the reagents. At lower EDTA concentrations (below 0.5 M), physicalcross linking of gelatin solutions was not observed and the gelatinbecame slightly viscous. Cross linking of gelatin solutions with mTGsolutions containing EDTA created strong flexible gels that wereadhesive.

Sodium citrate physically cross-linked 25% (w/w) gelatin solutions. Theformed gels were stable at 37° C. Sodium citrate formed highly flexible,strong gels that were not adhesive. 1M and 2M sodium citrate solutionsimmediately cross linked 25% (w/w) gelatin solutions with or withoutadditives. Sodium citrate solutions at concentrations ranging between0.5-0.75M immediately cross linked gelatin without additives. However,gelatin solutions containing urea became only viscous. According to theexperiments presented herein, using sodium citrate solutions atconcentrations ranging from 0.1M to 0.5M, did not result in physicalgelation. Above these concentrations, the higher the sodium citrateconcentration, the stronger the gel that was formed. The results suggestthe use of sodium citrate as a cross linking agent for gelatinsolutions, with or without the presence of mTG. The stability of theformed gels at 37° C. suggests that theses gels can be used for in situcrosslinking in the body cavity for applications such as surgicalsealing. Since sodium citrate does not appear to form adhesive gels withgelatin, the combination of sodium citrate and mTG may be preferred orconsidered. Gelatin solutions cross linked with mTG solutions containing0.5M sodium citrate form highly flexible, strong and adhesive gels.

Example 8 Effect of Calgon on mTG Crosslinking of Gelatin Solutions

As used herein, the term “Calgon” refers to amorphous sodiumpolyphosphate, such as for example sodium hexametaphosphate.

Materials

The following materials were used: 300 bloom, type A porcine gelatin[Sigma, St. Louis, Mo.], 98% urea [Alfa Aesar, Lancester], 0.1M SodiumAcetate buffer (pH 6.1) was prepared as previously described, 0.5MSodium Citrate dehydrate 99% [Alfa Aesar, Lancaster], Calcium Chloride97%, dried powder [Alfa Aesar, Lancester], Calgon [Global EnvironmentalSolutions, INC], 10% microbial Transglutaminase—ACTIVA-TG 10% (10%enzyme, 90% maltodextrin) [Ajinomoto, Japan].

A 4M urea stock solution in sodium acetate and a 4M stock solution ofcalcium chloride solution were prepared.

Methods

The following control and experimental solutions were prepared:Gelatin Solution A, 25% (w/w) gelatin with 4M ureaGelatin solution B, 25% (w/w) gelatin with 2M CaCl₂Gelatin solution C, 25% (w/w) gelatin with 2M urea 1M CaCl₂.Gelatin solution D, 25% (w/w) gelatin with 2M urea 2M CaCl₂.mTG solution 1, 0.5% mTG-5% (w/w) ACTIVA-TG 10% in 0.1M Na—Ac buffermTG solution 2, 0.5% mTG-5% (w/w) ACTIVA-TG 10% with 10% wt CalgonmTG solution 3, 0.5% mTG-5% (w/w) ACTIVA-TG 10% with 5% wt CalgonmTG solution 4, 1.0% mTG-10% (w/w) ACTIVA-TG 10% with 5% wt CalgonmTG solution 5, 2.0% mTG-20% (w/w) ACTIVA-TG 10% with 5% wt CalgonmTG solution 6, 1.0% mTG-10% (w/w) ACTIVA-TG 10% with 5% wt Calgon in0.1M sodium acetate buffer.mTG solution 7, 1.0% mTG-10% (w/w) ACTIVA-TG 10% with 5% wt Calgon in0.5M sodium citrate buffer.

Gel properties of gels formed by mixture of different gelatin solutionswith different mTG solutions were subjectively tested by thoroughlymixing 2 mL of gelatin solution with 1 mL of mTG solution. Gelation timewas then tracked by assessing gelation time as the time at which theentire gelatin mass formed a coherent solid gel.

Results

Initial tests of the use of Calgon proved to mildly decreasecross-linking time of control solutions with no calcium. However, noimproved mechanical properties were noted. Calgon resulted in thecreation of extremely adhesive and elastic gels but also proved toincrease significantly cross-linking time with solutions that didcontain calcium additives. Furthermore, it seems that Calgon worksbetter with lower concentrations as 5% wt Calgon solutions yieldedbetter and faster gels than equivalent 10% wt Calgon solutions.

TABLE 10 Gelation time and description of cross-linked gel 25% Gelatinsolution mTG Gelation Time Description of Cross-Linked Composition #plus: solution (min) Gel A1 A 1 1:10 Gel is rather adhesive. Some 4Murea 0.5% elasticity is noticed. Very weak mTG gel. in 0.1M After 10minutes gel is still NaAC somewhat adhesive, not very elastic andbecomes brittle. A2 A 2 0:50 Gel is slightly stronger than 4M urea 0.5%1 + A. Same adhesive property. mTG After 10 minutes still 10% somewhatadhesive and Calgon becomes more elastic than composition 1A. Becomesbrittle. B1 B 1 1:55 Formed gel is adhesive and 2M CaCl₂ 0.5% weak. mTGAfter 10 minutes still posses In 0.1M good adhesive properties, but NaACbecomes brittle in a similar way to composition 1A. B2 B 2 — After 10minutes, mixture of 2M CaCl₂ 0.5% the two solutions still remains mTG ina liquid state. Color of the 10% mixture is significantly other Calgonthan other above mixtures. After 50 minutes (no in- between examinationswere taken) the formed mixture posses great adhesive and elasticproperties although it is not yet solid (very high viscous state). B3 B3 — After 10 minutes, mixture of 2M CaCl₂ 0.5% the two solutions stillremains mTG in a liquid state. Color of the 5% mixture is significantlyother Calgon than other above mixtures. After 50 minutes (no in- betweenexaminations were taken) the formed gel posses great adhesive andelastic properties. It is also quite strong. B4 B 4 6:00 Formed gel isnot completely 2M CaCl₂ 1.0% solid (very high viscous state). mTG After20 minutes formed gel is 5% very strong, very adhesive and Calgon veryelastic. B5 B 5 2:00 Formed gel is not completely 2M CaCl₂ 2.0% solid(very high viscous state). mTG After 10 minutes the gel is very 5%adhesive, very elastic and Calgon strong. Gel becomes brittle only after20 minutes, and still remains very adhesive and elastic.

In further tests, Calgon helped to create extremely adhesive and elasticgels but also significantly increased cross-linking time in solutionsthat contained calcium additives. It should be noted that the mechanicalproperties (adhesion, elasticity, strength) of gels formed with Calgonwere retained for a long period of time (45+ minutes), and improved withtime progression. Use of 10% w/w mTG solutions with 5% wt calgon, eitherin 0.1M sodium acetate or 0.5M sodium citrate, resulted in a shortercross-linking time (faster reaction) for 25% w/w gelatin in 2M urea 1MCa when compared with 25% w/w gelatin in 2M urea 2M Ca. 25% w/w gelatinin 2M urea 2M Ca solution formed cross-linked gel faster with mTGsolutions in 0.1M Na—Ac compared to mTG solutions in 0.5M Na-Citrate.

TABLE 11 Gelation time and description of cross-linked gel 25% GelatinGelation solution mTG Time Composition # plus solution (min) Descriptionof Cross-Linked Gel A6 A 6 0:35 Form cross-linked gel is adhesive, 4Murea 1.0% elastic and weak. Possess low mTG flexibility. 5% After 10minutes gel becomes less Calgon adhesive, remains weak and not very 0.1Melastic and becomes quite brittle. NaAc B6 B 6 N/A No cross-linking wasobserved, even 2M CaCl₂ 1.0% after 45 minutes. mTG 5% Calgon 0.1M NaAcC6 C 6 3:20 Gel is very adhesive, very weak and in 2M urea 1.0% asemi-liquid state. 1M CaCl₂ mTG After 10 minutes gel remains very 5%much adhesive, becomes notably Calgon stronger, and is elastic. 0.1MAfter 20 minutes cross-linked gel NaAc remains very adhesive, strong andelastic though it is starts to become “brittle” (unlike solution B1, gelis torn apart when force is applied instead of break apart) After 30minutes no notably changes in the gel properties. After 45 minutes gelis still very adhesive and elastic. Although it is quite strong, it isalso “brittle”. D6 D 6 7:30 Formed gel is very adhesive and very 2M urea1.0% weak. The gel is less formed compared 2M CaCl₂ mTG to gel fromsolution B3 as it looks quite 5% liquid. Calgon After 10 minutes abetter formed gel is 0.1M achieved, although semi-liquid state NaAcstill remains. Gel is very adhesive, weak and “sticky”. After 20 minutesstill remains very weak semi-formed gel, with some elasticityproperties. A7 A 7 0:20 Gel is weak, somewhat adhesive. 4M urea 1.0%After 10 minutes gel becomes brittle. mTG 5% Calgon 0.5M citrate B7 B 7N/A No cross-linking was observed, even 2M CaCl₂ 1.0% after 45 minutes.mTG 5% Calgon 0.5M citrate C7 C 7 1:45 Cross-linked gel is elastic,flexible and 2M urea 1.0% very adhesive. Although the gel is not 1MCaCl₂ mTG very strong, it is much better than gel 5% achieved from 3Band is not in a semi- Calgon liquid state. 0.5M After 10 minutes gelremains very citrate adhesive. It is also possess good elasticity andflexibility properties. After 20 minutes gel remains very adhesive,elastic and flexible. Although it is quite strong, the gel is tear apartwhen force is applied (like in composition 3B). After 30 minutes gelappears to be stronger than before and still very adhesive, elastic andflexible. After 45 minutes remains very adhesive, quite flexible andelastic. Gel feels somewhat weaker. D7 D 7 13:10  Gel is extremely weakand it appears 2M urea 1.0% that besides being adhesive, possess no 2MCaCl₂ mTG other notably mechanical properties. 5% After 10 minutes, gelis very weak, soft Calgon and very adhesive. 0.5M After 20 minutes, gelis much stronger citrate and is very adhesive, elastics and flexible.After 30 minutes no notable changes in the gel properties. After 45minutes gel it appears that the gel become much stronger than before,and is more adhesive and elastic than formed gel from solution B3. Asbefore, gel is torn apart (and not “brittle”) when force is applied.

Example 9 Use of Urease to Reverse Sol-Gel Transition Point LoweringEffect of Urea

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), 98% urea (Alfa Aesar,Lancester), Sodium Acetate trihydrate (Sigma-Aldrich, St. Louis), AceticAcid 100% (Ridel-De Haen) Urease Type III: from Jack Beans(Sigma-Aldrich, St. Louis).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, and 4.5M Ureasolution were prepared.

25% w/w gelatin solution in 0.1M Sodium Acetate (Solution A) and 25%(w/w) Gelatin solution with 4.5M urea, 0.1M Sodium Acetate (Solution B)were prepared.

After homogenous solutions were achieved, half of the solution B wasmoved to another beaker (solution C) in which 0.125 g urease (5000units) was added and constant stirring was applied. Solutions A, B, andC were then transferred to a 22° C. environment.

Results

After 60 minutes at 22° C., it was found that while solution B remainedin viscous liquid form, solution C and A reverted to gel form.

These results indicate that urease reverses the sol-gel transition pointlowering effect of urea addition to a gelatin solution.

Example 10 Effect of Sorbitol on Crosslinked Gel Flexibility; Effect ofSorbitol on Accelerating mTG Crosslinking Reaction Materials

The following materials were used: Gelatin—300 bloom, type A porcinegelatin. [Sigma, St. Louis, Mo.], 98% urea [Alfa Aesar, Lancester], 0.1MSodium Acetate buffer (pH 6.1), Calcium chloride 97% [Alfa Aesar,Lancester], Sodium citrate dehydrate 99% [Alfa Aesar, Lancaster], Citricacid anhydrous [Frutarom, Israel], 10% microbialTransglutaminase—ACTIVA-TG 10% (10% enzyme, 90% maltodextrin)[Ajinomoto, Japan].

Methods

The following solutions were prepared: 4M urea solution in Na—Ac, 4 M ofCalcium solution, 2 M of Sodium Citrate solution was prepared, 0.5MSodium Citrate was prepared.

Gelatin Solution A—25% (w/w) gelatin with 4M urea.Gelatin solution B—25% (w/w) gelatin with 2M urea and 1M CamTG solution 1—0.5% w/w mTG solution (5% (w/w) ACTIVA-TG solution) with1:1 (w/w) (with respect to dry gelatin weight in gelatin solutions)sorbitol and 0.5M Na-CitratemTG solution 2—0.5% w/w mTG solution (5% (w/w) ACTIVA-TG solution) with2:1 (w/w) (with respect to dry gelatin weight in gelatin solutions)sorbitol and 0.5M Na-CitratemTG solution 3—0.5% w/w mTG solution (5% (w/w) ACTIVA-TG solution) with3:1 (w/w) (with respect to dry gelatin weight in gelatin solutions)sorbitol and 0.5M Na-Citrate.

Results

The experimental data in this experimental example confirmed thatsorbitol further enhances the flexibility of gels prepared with mTG insodium citrate buffer.

As illustrated in Tables 12-14, the use of sorbitol and sodium citratein mTG solutions proved to yield fairly flexible gels, both with 25%(w/w) gelatin in 4M urea and 25% (w/w) gelatin in 2M urea and 1M CaCl₂.Thus, it may be that sorbitol serves to enhance flexibility property aswell as to maintain mTG activity. It was also found that increasingsorbitol concentration leads to a faster cross-linking reaction.

TABLE 12 Cross-linking with 0.5% mTG (5% ACTIVA-TG 10%), 1:1 (w/w)sorbitol to gelatin ratio and 0.5M Na Gelation State at Time Solution24° C. (min) Description of Cross-Linked Gel A Low 0:50 Quite adhesivegel. Gel appears to be 25% viscous quite elastic, flexible and strong.gelatin After 10 minutes, gel remains fairly 4M urea adhesive, elasticand flexible although it becomes brittle as time progress. B Liquid 1:55The cross-linked gel is adhesive and 25% is more elastic and flexiblethan gel 1, gelatin although it appears to be quite weak. 2M urea After10 minutes gel remains 1M CaCl₂ adhesive and elastic and becomesstronger.

TABLE 13 Cross-linking with 0.5% mTG (5% ACTIVA-TG 10%), 2:1 (w/w)sorbitol to gelatin ratio and 0.5M Na State at Gelation Solution 24° C.Time (min) Description of Cross-Linked Gel A Liquid 0:35 Much moreadhesive gel than the 25% earlier gel (with 1:1 w/w sorbitol). gelatinQuite elastic and strong. After 4M urea 10 minutes remains quiteadhesive and flexible, but appears to be weaker and becomes brittle. BLiquid 1:20 Very adhesive. Quite elastic, 25% flexible and weak. After10 minutes gelatin still posses good adhesive properties. 2M urea Gel isquite elastic and flexible 1M CaCl₂ and fairly strong.

TABLE 14 Cross-linking with 0.5% mTG (5% ACTIVA-TG 10%), 3:1 (w/w)sorbitol to gelatin ratio and 0.5M sodium citrate State at GelationSolution 24° C. Time (min) Description of Cross-Linked Gel A Liquid 0:25Adhesive gel. Very weak at first, and 25% semi liquid (though clearlynoticed as gelatin gel). 4M urea After 10 minutes remains with the sameadhesive properties and gains some mechanical strength. Gel is moreelastic and flexibile. B Liquid 0:50 Very adhesive gel. Somewhat elastic25% and, just like gel 1, appears to be gelatin in a semi liquid stat.2M urea After 10 minutes remains very much 1M CaCl₂ adhesive and becomestronger, and more elastic and flexible. Hard to tell if this gel isbetter than the earlier test of 2 (with 2:1 w/w sorbitol).

Example 11 Gum Arabic, Guar Gum, PVA, PEG 6000 and Polyvinylpyrrolidone(PVP) as Plasticizers

Gum Arabic, Guar Gum, Polyvinyl Alcohol (PVA), Polyethylene Glycol (PEG)6000, and PVP are plasticizers that can act as spacers in a solutioninto which they are dissolved. This example demonstrates that theseplasticizers can be added to a crosslinker solution that is used tocrosslink a protein solution to improve the flexibility of thecrosslinked protein solution.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),ACTIVA TG (10% protein, 90% maltodextrin. Ajinomoto, Japan), SodiumCitrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Sodium Chloride (Frutarom, Israel), GumArabic (Sigma-Aldrich, St. Louis), PVA (Merck; Darmstadt, Germany),PEG6000 (Fluka; St. Louis, Mo.), Guar Gum (Sigma-Aldrich; St. Louis,Mo.), PVP in the form of Plasdone K-90 (ISP Technologies Inc.; TexasCity, Tex.).

The following stock solutions were prepared: 0.1M Na—Ac solution pH 6.0,0.4M Na-Citrate solution pH 6.0, 4.5M urea in 0.1 Na—Ac solution, 2MCaCl₂ stock solution in 0.1 M Na—Ac pH 6.0, 40% w/v Arabic gum solutionin WFI, 5% w/v Guar gum solution in WFI, 20% w/w PVP solution in WFI, 5%w/v PVA solution in WFI, 5% w/v Guar gum solution in WFI, 10% w/v PEG6000 in WFI.

Gelatin solutions of 12.5% w/w (solution A) and 25% w/w (solution B)were prepared in buffer of 3.8M Urea, 0.15M CaCl₂ and 0.1 M Na—Ac.

mTG powder solutions of 0.375% w/w (solution 1) and 0.5% w/w (solution2) were prepared by dissolving Activa TG (10% mTG, 90% maltodextrin) in0.4M Na-Citrate.

Methods

Aliquots of 0.375% mTG solution (solution 1) were mixed with each stockplasticizer solution at a volumetric ratio of 1:1. If the requiredconcentration of plasticizer was lower than half the stock solution, theplasticizer was diluted prior to mixing with the in TG solution. Foreach type of mTG-plasticizer solution, 10 mL of the solution wasthoroughly mixed with 20 mL of gelatin solution A and poured into a 100mL beaker.

The gelatin-mTG-plasticizer plug formed by the 30 mL of material in the100 mL beaker was removed from the beaker within 10 minutes followingmixture of the solutions. Each plug was then stored in physiologicalsaline for 24 hours at room temperature.

Following the 24 hour storage period, each plug was palpated by a blindtester who judged the flexibility of the plug on a scale of 1-3 (+, ++,or ++). The flexibility mark for each type of plug was recorded.

Results

The control gel (no plasticizer) was marked as the baseline flexibility(+) and the other plasticizer groups received marks indicating anoticeable increase in the flexibility of the gel, as indicated in thebelow table:

TABLE 15 Elasticity of formed gelatin gel as function of plasticizercomponent Condition of Gel Conc. Ratio after 24 hr in In mTGpolymer/gelatin Saline Control — — + — — + — — + Gum Arabic   5% 10.1%  ++  20% 40.1%   +++ Guar Gum 0.5% 1% ++ PVA 1.25%  2.5%   ++ 2.5% 5% ++PEG 6000 2.5% 5% ++   5% 10%  ++

Example 12 Effect of Concentration of an mTG Solution on Gelling TimeMaterials & Methods

An mTG solution was prepared by dissolving 10% (w/w) mTG powder into0.1M sodium acetate buffer. mTG used was ACTIVA WM (Ajinomoto, Japan),comprised of 99% maltodextrin and 1% protein. The volume of each mTGsolution was concentrated to the indicated factor using a 50% Daultrafiltration cartridge to remove the carrier materials and buffer.

Separately, gelatin solution containing 20% (w/w) type A, 300 bloomgelatin in 0.1M sodium acetate buffer of pH 6.0 was prepared.

After mTG solutions were concentrated, aliquot of each mTG solution wereadded to aliquots of gelatin solution at a volumetric ratio of 1:2, mTGsolution: gelatin solution. This mixture was then thoroughly mixed.Mixed composition was subject to inversion in a tube every 30 secondsand gelling time was defined as time at which composition ceased toflow.

Results

Table 16 shows the effect of concentration of mTG solution on gellingtime.

TABLE 16 Effect of concentration of mTG solution on gelling time mTGConcentration factor Gelling time (min) No treatment — 6 After membrane— 7 pretreatment with 0.45μ After concentration 1.5 4 Afterconcentration 2 3 After concentration 3 2

Example 13 Microbial Transglutaminase (mTG) Purification Process

This example relates to a microbial transglutaminase (mTG) purificationprocess that purifies a mTG product, which in this non-limiting exampleis food-grade, to produce an mTG composition with specific activity >25enzyme units per milligram, >95% electrophoretic purity, <5 endotoxinunits per gram, and <10 CFU/g.

Food grade mTG product (Activa™ TG; Ajinomoto, Japan) was used as astarting raw material. The initial characteristics of this mTG productwere as shown in Table 17:

TABLE 17 mTG initial characteristics Tests Specifications M.W.(SDS- 38kDa ± 2 kDa Coomassie) SDS-PAGE >95% (Coomassie) Specific activity 11.4U/mg, Bradford

This food grade product was processed with the below-describedpurification process scheme:

TABLE 18 Process scheme for mTG purification: Step Details Dissolution1700 g of mTG powder at concentration of 7.5% w/w in buffer (50 mM NaAcpH 5.5), high stirring rate for 1 hour at room temperature. FiltrationCoarse filtration with medical bandage to remove aggregates.Clarification Ultrafiltration (UF) with pore size of 0.65μ; Filtrate wascollected SP-FF 24 ml column chromatography Equilibration: 50 mM NaAc pH5.5 Load: post clarification fraction Wash: 50 mM NaAc, 50 mM NaCl pH5.5 Elution: 50 mM NaAc, 150 mM NaCl pH 5.5 Concentration 10 kDa cutoffand dialysis Buffer change to 0.2M citrate pH 6.0The resulting, purified mTG solution was as follows, as shown in Table19A:

TABLE 19A Tests Specifications M.W.(SDS- 38 kDa ± 2 kDa Coomassie)SDS-PAGE >95% (Coomassie) Specific activity 26.3 U/mg LAL-endotoxins<0.15 EU/g mTG solution AMC <10 CFU/ml mTG solution

FIG. 4 shows the results of gel electrophoresis of the purified material(lanes 2-8). Each lane was loaded with a different amount of purifiedmTG, ranging from 1-20 μg, as shown in Table 19B:

TABLE 19B μg Adj. Vol. Lane loaded ODu * mm2 2 1 99.82786 3 2 150.1723 44 216.5421 5 6 286.3214 6 8 316.2235 7 10 366.3012 8 20 486.6132

The molecular weight standards are shown in lane 1, with molecularweights as given. Purified mTG is represented by the major band at about38 kd. The bands for 6 micrograms of protein (lane 5) underwentdensitometric analysis since densitometric linearity was not calculatedat loading amounts above 6 μg. The results are shown in Table 20 below.

TABLE 20 Densitometric analysis results Adj. vol Net. % of Band ODu *mm2 Vol. total U1 338.288622 334.749 95.9354 U2 13.697427 5.346581.53227 U3 12.8509144 4.97192 1.4249 U4 15.7683192 2.99658 0.85879 U59.03115586 0.86754 0.24863

The results in Table 20 above show that main band density contains˜95.9% of total proteins in the sample. This purification processdemonstrates that it is possible to purify food grade mTG into a mTGcomposition that is more suitable for medical use.

Example 14 Use of Alginate Ester, Gum Arabic, Carboxymethyl Cellulose(CMC), Xanthan Gum, Guar Gum, PVP to Increase Viscosity of CrosslinkerMaterial Solution

Alginate Ester, Gum Arabic, Carboxymethyl cellulose (CMC), Xanthan Gum,Guar Gum, and PVP are well known viscofiers that increase the visocityof solution into which they are dissolved. This example demonstratesthat these viscofiers can be added to a crosslinker solution that isused to crosslink a protein solution without inhibiting the crosslinkingrate that results in gelation of the protein solution by more than 50%.Even more surprisingly, some of these viscofiers accelerate the gelationspeed.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),ACTIVA TG (10% protein, 90% maltodextrin. Ajinomoto, Japan), SodiumCitrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Sodium Chloride (Frutarom, Israel), AlginateEster (Sigma-Aldrich; St. Louis, Mo.), Gum Arabic (Sigma-Aldrich, St.Louis), Carboxymethyl Cellulose (high and medium viscosity,Sigma-Aldrich; St. Louis, Mo.), Xanthan gum (Sigma-Aldrich, St. Louis),Guar Gum (Sigma-Aldrich; St. Louis, Mo.), Plasdone K-90 (ISPTechnologies Inc.; Texas City, Tex.).

The following stock solutions were prepared: 0.1M Na—Ac solution pH 6.0,0.4M Na-Citrate solution pH 6.0, 4.5M urea in 0.1 Na—Ac solution, 2MCaCl₂ stock solution in 0.1M Na—Ac pH 6.0, 5% w/v Alginate Estersolution in water for injection (WFI), 40% w/v Arabic gum solution inWFI, 4.2% w/v medium viscosity CMC solution in WFI, 2.5% w/v highviscosity CMC solution in WFI, 1.8% w/v Xanthan gum solution in WFI, 5%w/v Guar gum solution in WFI, 20% w/w Plasdone solution in WFI.

Gelatin solutions of 12.5% w/w (solution A) and 25% w/w (solution B)were prepared in buffer of 3.8M Urea, 0.15M CaCl₂ and 0.1M Na—Ac.

mTG powder solutions of 3.75% w/w (solution 1) and 5% w/w (solution 2)were prepared by dissolving Activa TG in 0.4M Na-Citrate.

Methods

Aliquots of 3.75% mTG solution (solution 1) was mixed with each stockviscofier solution at a volumetric ratio of 1:1. If the requiredconcentration of viscofier was lower than half the stock solution, theviscofier was diluted prior to mixing with the mTG solution. EachmTG-viscofier solution was qualitatively assessed for viscosity bymanual stirring.

For each type of mTG-viscofier solution, 10 mL of the solution wasthoroughly mixed with 20 mL of gelatin solution A and poured into a 100mL beaker. The viscosity increase of the mixed solution was then trackedusing a DV-II+ Pro Viscometer (Brookfield; Middleboro, Mass.) using at-bar type spindle moving along a helical path at a speed of 0.5 RPM.This experiment was repeated 3 times for each type of mTG-viscofiersolution.

Crosslinking rate was defined according to the amount of time thatgelatin-mTG-viscofier solution took to achieve viscosity of 9×10⁶ cP.The average crosslinking rate of each gelatin-mTG-viscofier compositionwas then compared to the average of a control gelatin-mTG compositioncontaining no viscofiers to determine percentage of crosslinkinginhibition.

Results

Viscosity of mTG solutions with plasdone, xanthan gum, CMC, gum Arabic,and alginate ester were observed to be noticeably more viscous that mTGsolution without addition of viscofier.

Relative crosslinking rates of gelatin-mTG solutions with differentviscofiers can be seen in FIG. 5, which shows the relative cross linkingrate of different plasticizer solutions compared to control. Crosslinking was measured quantitatively using viscometery. Percentage valuesin column title refer to concentration of plasticizer in gel.

No viscofier inhibited reaction by more than 30%. Plasdone, xanthan gum,and high viscosity CMC accelerated the crosslinking rate.

Example 15 Glutamate Improves Tissue Response to a mTG-CrosslinkedGelatin Composition Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Sigma-Aldrich,St. Louis), Sodium Citrate (Sigma-Aldrich, St. Louis), Citric AcidMonohydrate (Sigma-Aldrich, St. Louis), L-Glutamic Acid (Sigma-Aldrich,St. Louis), microbial transglutaminase—ACTIVA-TG (10% enzyme, 90%maltodextrin) (Ajinomoto, Japan), 8 Sprague-Dawley (SD) rats.

Two gelatin solutions were prepared:

-   -   1) 25% (w/w) Gelatin solution in 4.5M Urea, 0.1M Sodium Acetate        buffer (solution A).    -   2) Above, to which L-glutamate was added at a concentration of        1.2 g/100 mL of gelatin solution (solution B).        A 7.5% (w/w) Activa TG solution in 0.2M Na-Citrate (mTG        solution) was also prepared.

Methods

Prior to each implantation, one gelatin solution was mixed with mTGsolution at a volumetric ratio 2:1. 0.1 mL of the mixed gelatin-mTGcomposition was then immediately implanted at 4 separate subcutaneoussites in a SD rat. This was repeated in 8 rats, 4 rats with gelatinsolution A and 4 rats with gelatin solution B (glutamate).

After 14 days, the rats were sacrificed. Lesions from the implantationsites were removed and histopathological evaluations were performed.

All tissues were collected from all animals during the respectivescheduled necropsy sessions and fixed in 10% neutral buffered formalin(approximately 4% formaldehyde solution) for at least 48-hr fixationperiod prior to their shipment to the testing laboratory.

Slide preparation followed by histopathological examination wasperformed for tissues listed in the study protocol. Tissues weretrimmed, embedded in paraffin, sectioned at approximately 5 micronsthickness and stained with Hematoxylin & Eosin (H&E).

General assessment and scoring for each of the groups sacrificed after14 days was performed individually. Total scoring values are relativeand contain both chronic and acute inflammation scores.

Results

Histopathological scoring for rats sacrifice after 14 day implantationof each material (values are average of 4 animals). Results are shown inTables 21 and 22.

TABLE 21 Gelatin Solution A + mTG: sacrifice after 14 days Animal Number13 14 16 17 Site number a b c d a b c D a b c d a b c d Polymorhonuclearcells* 3 3 3 3 Eosinophils* 3 3 3 3 Lymphocytes* 1 1 1 1 Plasma cells* 11 1 1 Macrophages* 2 2 2 2 Giant cells* 1 1 1 1 Necrosis 0 0 0 0 Totalper site 11 11 11 11 SUBTOTAL I (×2) 88 88 88 88 Fiboplasia 2 2 2 2Fibrosis 3 3 3 3 Total per site 5 5 5 5 SUBTOTAL II 20 20 20 20 TOTAL[SUB-TOTAL I + II] 108 108 108 108 GROUP TOTAL 432 GROUP AVERAGE** 27

TABLE 22 Gelatin Solution B (glutamate) + mTG: sacrifice after 14 daysAnimal Number 18 19 20 21 Site number a b c d a b c D a + b c d a b c dPolymorhonuclear cells* 2 2 2 2 Eosinophils* 2 2 2 2 Lymphocytes* 1 1 11 Plasma cells* 1 1 1 1 Macrophages* 3 3 2 2 Giant cells* 1 1 1 1Necrosis 0 0 0 0 Total per site 10 10 9 9 SUBTOTAL I (×2) 80 80 72 72Fiboplasia 2 2 2 2 Fibrosis 3 3 3 Total per site 5 5 5 5 SUBTOTAL II 2020 20 20 TOTAL [SUB-TOTAL I + II] 100 100 92 92 GROUP TOTAL 384 GROUPAVERAGE** 24

These results indicate that under otherwise identical conditions, theaddition of glutamate to an implanted crosslinked gelatin compositionresulted in a lower level of inflammatory reaction.

Example 16 The Effect of Proline and Trehalose on Restoring the PhysicalGelation of a Gelatin Solution with a Chaotrope-Lowered Sol-GelTransition Point Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Proline (Sigma-Aldrich, St. Louis), Trehalose dihydrate (Sigma-Aldrich,St. Louis).

Methods

Stock solutions of 0.1 M Sodium Acetate buffer pH 6.0, buffer pH 6.0,4.5M Urea, 2M CaCl2, were prepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(Control solution), 25% (w/w) Gelatin solution in 1M Proline, 3.8M Urea,0.15M Ca, 0.1M Sodium Acetate (Solution A), 25% (w/w) Gelatin solutionin 1.5M Proline, 3.8M Urea, 0.15M Ca, 0.1 M Sodium Acetate (Solution B);25% (w/w) Gelatin solution in 0.5M Proline, 0.4M Trehalose 3.8M Urea,0.15M Ca, 0.1M Sodium Acetate (Solution C); 25% (w/w) Gelatin solution1.5M Proline, 0.4M Trehalose 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(Solution D); were prepared. All solutions were heated to 50° C. underconstant stirring to achieve homogenous solutions.

After solutions were obtained, all solutions were moved to athermostatic bath set to 25° C. After 60 minutes, physical state of eachsolution was checked by palpating the solution. Solutions were thenmoved to a 22° C. and physical state was checked again after another 60minutes.

Results

The control solution remained, as expected, in mildly viscous liquidform at 25° C. Solution A was very highly viscous. Solutions B, C and Dwere completely gelled after 60 minutes.

After the additional 60 minutes at 22° C., Solution A formed a solid gelas well.

Control gelatin solution did not form a gel at 22° C. or 25° C.

These observations indicate that the addition of Proline leads to ahigher transition point of gelatin solutions in comparison to controlsolutions. The results also indicate that there is a synergistic effectbetween Proline and Trehalose when combined together, in comparison toProline alone, as 0.5M Proline with 0.4M Trehalose caused gelation at25° C. whereas 1M Proline alone did not.

Example 17 Effect of Proline and Glutamate (Kosmotropes) to Increase theElasticity of mTG-Crosslinked Gelatin Gels

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), L-Proline 99% (Sigma, St. Louis, Mo.),L-Glutamic acid (Glutamate), non animal source (Sigma, St. Louis, Mo.),10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme, 90%maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1 M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCl₂, were prepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(solution A), 0.25% (w/w) mTG in 0.2M Na-Citrate (mTG control), 0.25%(w/w) mTG with 0.04 g/ml Glutamate in 0.2M Na-Citrate (mTG 1) and 0.25%(w/w) mTG 2.5M Proline in 0.2M Na-Citrate (mTG 2) were prepared.

Tensile testing was then performed on crosslinked gels formed by mixingaliquots of each of the above gelatin solutions with aliquots of mTGsolution.

For each test, 6 ml of gelatin solution were mixed with 3 ml of mTGsolution. The resulting mixture was applied to dog bone shaped molds,with 2 mL in each mold. The effective testing cross-sectional area ofthe gels formed in these molds was 12 mm by 1.7 mm. Molds containinggels were incubated at 370° C. for 10 min. After incubation, the moldswere covered in saline and the formed gels were extracted from themolds.

For the testing of each gel, the tabs on either end of the dogboneshaped gel were clamped into a Model 3343 Single Column MaterialsTesting System (Instron™; Norwood, Mass.). The top tab was then pulledupwards at a rate of 0.5 mm/s, resulting in the creation of tensileforce on the gel dogbone. Tension of sample was continued until failurewas observed. Bluehill 2 Materials Testing Software (Instron™; Norwood,Mass.) was used to analyze results and calculate material propertiesincluding elastic modulus, peak stress, and strain to break.

Results

The material testing results indicate that both Proline and Glutamatecan be used to increase the elasticity of a mTG-crosslinked gelatincomposition, as shown in Table 23.

TABLE 23 Proline and Glutamate Increase the Elasticity of theComposition Average solution A reacted Average Tensile Stress AverageTensile with Modulus, kPa at Break, kPa Strain at Break, % mTG control79 50 62 mTG 1 74 58 84 (glutamate) mTG 2 (proline) 60 44 75

Example 18 Effect of Surfactants Tween 20 and Tween 80 on Increasing theElasticity of Crosslinked Protein Composition

This Example relates to the effect of surfactants when used above theirCMC (critical micelle concentrations). As a non-limiting example, twomembers of the “Tween” family are given. Tween™ hydrophilic surfactants(Polysorbates) are a family of PEG sorbitan esters(polyoxyethylene-sorbitan-fatty acid esters), for example mono- andtri-lauryl, palmityl, stearyl and oleyl esters of the type known andcommercially available under the trade name Tween™ (Fiedler, H. P.,“Lexikon der Hilfsstoffe fur Pharmazie, Kosmetic and AngrenzendeGebiete”, Editio Cantor. D-7960 Aulendorf, 3rd edition, 1989, pages1300-1304). Tween™ 20 (polyoxyethylene(20)sorbitan monolaurate) has anHLB of 16.7. Other types of Tween™ surfactants may also be useful forthe compositions of at least some embodiments of the present invention.

Tween™ surfactants are soluble in water but not in oil. The chemicalstructure of this family of surfactants features one, two or three shortPEG chains, generally of about 5 to 20 ethylene glycol units, connectedby an ester bond to sorbitan. These surfactants are produced by variouscompanies (Croda, ICI, Sandoz, Mazer, Atlas) and may appear undervarious trade names, besides Tween™: Sorlate™, Monitan™, Crillet™ and soforth. Members of this family which are polysorbates 20, 21, 0, 60, 61,65, 80 and 85 are preferred for this embodiment of the presentinvention.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Tween 20 (Sigma, St. Louis, Mo.), Tween 80(Sigma, St. Louis, Mo.), 10% microbial Transglutaminase—ACTIVA-TG 10%(10% enzyme, 90% maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1 M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCl₂, were prepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(solution A), 25% (w/w) Gelatin solution with 0.1% or 1% Tween 20 in3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate (solutions B, C respectively),25% (w/w) Gelatin solution with 0.1% or 1% Tween 80 in 3.8M Urea, 0.15MCa, 0.1M Sodium Acetate (solutions D, E respectively) and 0.25% (w/w)solution food-grade mTG in 0.2M Na-Citrate (solution 1) were prepared.

Tensile testing was then performed on crosslinked gels formed by mixingaliquots of each of the above gelatin solutions with aliquots of mTGsolution.

For each test, 6 ml of gelatin solution were mixed with 3 ml of mTGsolution. The resulting mixture was applied to dog bone shaped molds,with 2 mL in each mold. The effective testing cross-sectional area ofthe gels formed in these molds was 12 mm by 1.7 mm. Molds containinggels were incubated at 37 C for 10 min. After incubation, the molds werecovered, in saline and the formed gels were extracted from the molds.

For the testing of each gel, the tabs on either end of the dogboneshaped gel were clamped into a Model 3343 Single Column MaterialsTesting System (Instron™; Norwood, Mass.). The top tab was then pulledupwards at a rate of 0.5 mm/s, resulting in the creation of tensileforce on the gel dogbone. Tension of sample was continued until failurewas observed. Bluehill 2 Materials Testing Software (Instron™; Norwood,Mass.) was used to analyze results and calculate material propertiesincluding elastic modulus, peak stress, and strain to break.

Results

The material testing results indicate that both Tween20 and Tween80 areuseful for increasing the elasticity (strain to break) of thecrosslinked gelatin gels and that this effect is concentrationdependant, as shown in Table 24.

TABLE 24 Effect of Tween Surfactants on Elasticity Solution A Solution BSolution C Solution D Solution E Elastic Modulus (kPa) Average 103.16102.87 81.62 92.38 96.56 StdDev 18.10 8.68 14.22 13.26 10.74 Stress atbreak (kPa) Average 57.67 54.07 68.14 63.95 74.01 StdDev 28.94 12.5712.11 6.57 9.29 Strain at break (%) Average 57.17 54.86 89.24 73.3679.10 StdDev 25.14 14.26 5.03 15.80 8.28

Example 19 Use of Cystamine, Cysteine and Melanin as mTG Inhibitors

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Cystamine dihydrochloride 98%(Sigma-Aldrich, St. Louis), L-Cystein 97% (Sigma-Aldrich, St. Louis),Melanin (Sigma-Aldrich, St. Louis) 10% microbialTransglutaminase—ACTIVA-TG 10% (10% enzyme, 90% maltodextrin)(Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, were prepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(control solution), 25% (w/w) Gelatin 0.1% w/v Cystamine solution in3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate (solution A), 25% (w/w) Gelatin10% w/v Cystein solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(solution B), 0.75% (w/w) mTG in 0.2M Na-Citrate (solution 1), 0.25%(w/w) mTG in 0.2M Na-Citrate (solution 2), 0.25% (w/w) mTG in 0.2MNa-Citrate with 2 mg/ml Melanin (solution 3), 0.25% (w/w) mTG in 0.2MNa-Citrate with 10 mg/ml Melanin (solution 4) were prepared.

Gelatin solutions containing Cystamin and Cystein (A, B) were tested byviscometer with mTG solution 1.

mTG solutions containing Melanin (3, 4) were tested in a qualitativemanner with control solution.

Viscometer Tests

For each viscometry test, 20 mL of gelatin solution was mixed with 10 mLof mTG solution in a 50 mL beaker. The viscosity of the mixedgelatin-mTG solution was then tracked as it underwent gelation.Different test groups were compared by recording the time required foreach test group to achieve 30% and 90% of the maximum viscosity able tobe recorded by the viscometer at the specific speed and with thespecific spindle used for that test.

In this experiment, a DV II+ PRO Digital Viscometer (BrookfieldEngineering, Middleboro, Mass.) was used with a T-E 95 “t-bar” spindle.A helipath viscometer stand was used to maintain vertical movement ofthe spindle over the course of the viscometer test. The helipath movedalong a 1 cm path. The viscometer readings were outputted by theviscometer and read using HyperTerminal software at a rate of 1 readingper second. The rotational speed of the spindle for the viscometry testwas 0.5 rpm. The maximum recordable viscosity at this speed with the T-E95 spindle was 10×106 cP, meaning that the 30% point was equivalent to3×106 cP and the 90% point was equivalent to 9×106 cP.

The beaker was submerged in a 37° C. water bath for the entire extent ofthe viscometer test. Average temperature within the beaker also recordedthroughout the test to ensure consistency between test groups.

Qualitative Crosslinking Test

Gelatin and mTG solutions were mixed in 2:1 ratio, then moved to a 37 Cincubator and crosslinking time was defined when an apparent gelationwas detected.

Results

Cystamin and Cystein Inhibition Results:

FIG. 6 shows results of gelatin solutions control, A and B. As can beseen, addition of the Cystamine to the gelatin solution increasedcrosslinking time of the matrix by about 40%. Addition of Cysteinresulted an increase of about 28% in the average crosslinking time.These results demonstrate mTG inhibition by addition of Cystamine orCystein to the gelatin solution.

When examined, crosslinked gels from solutions containing Cystamine orCystein appeared to be more flexible than the equivalent crosslinked gelwith no Cystamine or Cystein additives.

Melanin Results:

Table 25 describes the results of crosslinking time with regard to theeffect of Melanin. When mTG was used with Melanin, crosslinking timeincreased significantly. Increasing Melanin concentration increasedcrosslinking time. This finding demonstrates mTG inhibition by additionof Melanin.

TABLE 25 Effect of Melanin on crosslinking time Composition ofcrosslinked gel Crosslinking time, min Control gelatin solution + mTG0:40 solution 2 (no melanin) Control gelatin solution + mTG 3:00solution 3 (2 mg/ml melanin) Control gelatin solution + mTG 6:00solution 4 (10 mg/ml melanin)

Example 20 Effect of PEG-Amine on the Kinetics of a Gelatin CrosslinkingReaction and the Elasticity of a Crosslinked Gelatin Composition

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), PEG-Amine MW 5000 (Nof Corporation, Japan),PEG 6000 (Fluka, Switzerland), microbial transglutaminase ACTIVA-TG (10%enzyme, 90% maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, were prepared.

25% (w/w) Gelatin in 3.8M Urea, 0.15M CaCl2, 0.1M Sodium Acetate(control solution), 25% (w/w) Gelatin with 5% w/v PEG-amine in 3.8MUrea, 0.15M CaCl2, 0.1M Sodium Acetate (Solution A), 25% (w/w) Gelatinwith 10% PEG-amine in 3.8M Urea, 0.15M CaCl2, 0.1M Sodium Acetate(Solution B), 25% (w/w) Gelatin with 20% PEG-amine in 3.8M Urea, 0.1-5MCaCl2, 0.1M Sodium Acetate (Solution C), 25% (w/w) Gelatin with 20% PEG6000 in 3.8M Urea, 0.15M CaCl2, 0.1M Sodium Acetate (Solution D), 0.25%(w/w) mTG in 0.2M Na-Citrate (mTG solution) were prepared.

Solutions were examined using Qualitative and Elasticity tests.

Qualitative Crosslinking Test

Gelatin solutions A, B, and C were each mixed with the mTG solution at a2:1 volumetric ratio in glass tubes, then moved to a 37° C. shakingincubator. Gelation time was defined as the time at which flow of theliquid solution was observed to cease.

Elasticity Test

The control solution and Solution B crosslinked with mTG solution wereexamined for changes in elasticity over time.

For each elasticity test, 6 mL of gelatin solution were mixed with 3 mlof mTG solution. The resulting mixture was applied to dog-bone mold, 2mL in each mold. Molds were incubated at 37° C. for 10 min. After theincubation, molds were covered in saline and gelatin-mTG compositionextracted from the mold.

The specimens were then incubated in saline at 37° C., or examinedimmediately. Thickness of gelatin-mTG composition removed from gel wasmeasured using a caliper.

For the testing of each gel, the tabs on either end of the dogboneshaped gel were clamped into a Model 3343 Single Column MaterialsTesting System (Instron™; Norwood, Mass.). The top tab was then pulledupwards at a rate of 0.5 mm/s, resulting in the creation of tensileforce on the gel dogbone. Tension of sample was continued until failurewas observed. Bluehill 2 Materials Testing Software (Instron™; Norwood,Mass.) was used to analyze results and calculate material propertiesincluding elastic modulus, peak stress, and strain to break.

Results

Qualitative Crosslinking Test

Table 27 displays the results for the qualitative crosslinking test,showing that increasing amounts of PEG-amine decreases the time togelation.

TABLE 27 results of crosslinking Gelatin Solution Gelation time [min]Solution A 3:50 Solution B 2:00 Solution C 1:20 Control Solution 4:40

Elasticity Test

Table 28 displays the average results for the elasticity tests; resultsare for 2 hr incubation in saline at 37° C. The results show thatPEG-amine can increase the elasticity of the gelled composition.

TABLE 28 Elasticity Test Results Tensile Stress at Tensile Strain atGelatin Solution Modulus (kPa) Break (kPa) Break (%) Control 90.50 46.2851.23 Solution B 48.06 35.71 80.18 Solution D 55.03 30.13 57.00

Overall, these results indicate that the inclusion of PEG-Amine canaffect the gelation kinetics of mTG-crosslinking of a gelatin solution;and the inclusion of PEG-Amine can increase the elasticity of acrosslinked gelatin composition. Another non-limiting example of asuitable PEG derivative capable of covalently binding to gelatin isPVA-amine, which is also encompassed by this embodiment of the presentinvention.

Example 21 Inhibition of Carbamylation

This Example shows that glycine inhibits carbamylation in a gelatinsolution containing urea in a dose dependant manner without inhibitingmTG cross-linking afterwards. Histidine can similarly be used to inhibitcarbamylation.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Glycine, non animal source (Sigma, St.Louis, Mo.), 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme,90% maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, were prepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(control solution), 25% (w/w) Gelatin 0.1M Histidine solution in 3.8MUrea, 0.15M Ca, 0.1M Sodium Acetate (solution A), 25% (w/w) Gelatin 0.1MGlycine solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate (solutionB), 25% (w/w) Gelatin 0.4M Glycine solution in 3.8M Urea, 0.15M Ca, 0.1MSodium Acetate (solution C), 25% (w/w) Gelatin 0.5M Glycine solution in3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate (solution D), 25% (w/w) Gelatin0.7M Glycine solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(solution E), 25% (w/w) Gelatin 0.9M Glycine solution in 3.8M Urea,0.15M Ca, 0.1M Sodium Acetate (solution F), 25% (w/w) Gelatin 1M Glycinesolution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate (solution G), 0.25%(w/w) mTG in 0.2M Na-Citrate (solution 1), 0.75% (w/w) mTG in 0.2MNa-Citrate (solution 2) were prepared.

Results

FIGS. 7A, 7B and 7C summarize results of gelatin solutions with variousGlycine and Histidine additives, functioning as Carbamylationinhibitors, which were crosslinked and tested by viscometer afterdifferent incubation times at high temperature (50° C.). All figurespresent values of both 30% and 90% of overall crosslinking time.

FIG. 7A presents normalized crosslinking time values of gelatinsolutions with and without Glycine or Histidine additives (0.1 M each)after one day of incubation at 50° C. Values are normalized based on ancontrol solution with no additives which was stored at 4 C for one day,therefore value of 100% defines the time needed to achieve crosslinkingtime as the gelatin control solution. FIG. 7A shows the normalized crosslinking time of gelatin control, solution A and solution B afterincubation at 50 C for one day. The cones show the time to torque 30%while the cylinders show the time to torque of 90%. The data are shownin pairs: the first two relate to control solution alone, the secondrelate to solution A and the third pair relates to solution B.

Results from FIG. 7A indicate that after a day of incubation, bothgelatin solution with 0.1M Histidine (A) and gelatin solution with noadditives which was incubated at 50° C. (control) took 38%-40% longer toachieve viscosity levels compared to control which was stored at 4° C.However, use of 0.1M Glycine (B) reduced the level of reactioninhibition, with crosslinking reaction delay being limited to 14-17%.

FIG. 7B presents normalized crosslinking time values of gelatinsolutions with and without Glycine or Histidine additives after two daysof incubation at 50° C. The cones show the time to torque 30% while thecylinders show the time to torque of 90%. The data are shown in pairs:the first two relate to solution B alone, the second relate to solutionC, the third pair relates to solution F and the fourth pair relates tosolution F. Values were normalized based on an gelatin control solutionwith no additives which was also incubated at 50° C. for two days,therefore 100% of crosslinking time is a time needed to achievecrosslinking time as the control incubated solution.

Results from FIG. 7B indicate that gelatin solution with highest Glycineconcentration, 0.9M (solution F), led to the best crosslinking time. Thereaction was about 22% faster than the control solution, while Glycineconcentration of 0.1M (solution B) led to a reaction only to 5-6% fasterreaction time than control. Use of gelatin solution with 0.1M Histidine(solution A) led to similar crosslinking time as in the gelatin solutionwith 0.9M Glycine (F), with the crosslinking time was 18% faster thanthe control solution.

FIG. 7C presents crosslinking time values of gelatin solutions withGlycine after two weeks of incubation at 50 C, and crosslinking timevalues of gelatin control solution stored at 4 C for two weeks. Thecones show the time to torque 30% while the cylinders show the time totorque of 90%. The data are shown in pairs: the first two relate tocontrol solution alone, the second relate to solution D, the third pairrelates to solution E, the fourth pair relates to solution F and thefifth pair relates to solution G.

Results from FIG. 7C indicate that use of gelatin solution with thehighest Glycine concentration does not necessarily lead to the bestresults, as crosslinking time of gelatin solution with 0.9M Glycine (F)was by 50% lower than crosslinking time achieved with gelatin solutionwith 1M Glycine (G). Also, it appears 0.9M Glycine was the optimumconcentration as 0.7M Glycine (E) achieved crosslinking time higher byabout 50% than gelatin solution with 0.9M Glycine. Results alsoindicated that after more than 23 minutes, no crosslinking was observedwith gelatin solution with no additives, an indication of thedeterioration of the solution.

Overall, these results indicate that both glycine and histidine caninhibit carbamylation reactions in gelatin. However, the specificconcentrations and choice of substances is dependant on the conditionsof cyanate production in solutions.

Example 22 Cyanate Addition for Partially Crosslinking of GelatinSolutions Containing No Urea

This Example shows that the presence of sodium cyanate results in aninhibitory effect, confirming that urea breakdown is responsible forinhibition of mTG crosslinking.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Sodium Acetate trihydrate(Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ride)-De Haen), SodiumCitrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Sodium Cyanate 96% (Sigma-Aldrich, St.Louis), 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme, 90%maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0 and 0.2M SodiumCitrate buffer pH 6.0 were prepared.

25% (w/w) Gelatin solution in 0.1M Sodium Acetate (control solution),25% (w/w) Gelatin+0.1M Sodium Cyanate in 0.1M Sodium Acetate (solutionA) and 0.25% (w/w) mTG in 0.2M Na-Citrate (solution 1) were prepared.

Viscometer Tests

Viscometer tests were carried out as described in Example 19.

Results

FIG. 8 summarizes results of control solutions with and without Cyanateadditives which were crosslinked and tested by viscometer afterdifferent incubation times of high temperature (50 C). The cones showthe time to torque 30% while the cylinders show the time to torque of90% (ie cross linking time). The data are shown in pairs: the first tworelate to the control solution, while the second relate to solution A.Values of crosslinking time are normalized based on control solutionwith no additives which was stored at 4 C for one day. Therefore, valueof 100% is the time needed to achieve crosslinking of that solution.

Results indicate that while the control solution with incubation of 50 Cshowed a slightly increased crosslinking time (by about 10%), thesolution which also contained 0.1M Cyanate (solution A) increasedsignificantly its crosslinking time by more than 76%. This findingdemonstrates that addition of Cyanate anions, either with directaddition or by decomposing urea, can significantly deterioratecrosslinking time of gelatin solutions and has a significant inhibitoryeffect.

Example 23 A Gelatin Solution can be Completely Succinylated Such thatthe Gelatin is No Longer a Substrate for mTG Crosslinking

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Succinic anhydride(Sigma-Aldrich, St. Louis), Sodium hydroxide (Ridel-De Haen), SodiumBicarbonate (Frutarum, Israel), Sodium Citrate (Sigma-Aldrich, St.Louis), 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme, 90%maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Bicarbonate buffer pH 8.0, 4M Sodium hydroxideand 0.2M Na-Citrate were prepared.

1% (w/w) Gelatin solution in 0.1M Bicarbonate buffer was prepared(solution A). The solution was heated to temperature of 37 C whileconstant stirring was applied. Also, 0.75% (w/w) mTG in 0.2M Na-Citrate(mTG solution) was prepared.

After a homogenous steady state was achieved, succinic anhydride wasadded to the solution in powdered form. The solution's pH was monitoredconstantly and was kept at pH=8 by addition of 4M Sodium hydroxide. Whenno more pH changes were observed, the solution was placed in dialysisbag and submerged in distilled water for 24 hr.

The resultant solution was concentrated using an Ultrafiltration processwith a 30 kDa membrane cut-off. During the process intake pressure didnot exceed 1.5 bar.

The solution's concentration was determined using the solution'sabsorbance at 280 nm (Solution A). A corresponding solution was prepared(Solution B) of unsuccinylated gelatin at the same concentration assolution A.

2.5 ml of mTG solution was mixed with 5 ml of solutions A and B,respectively, and placed at 40 C.

Results

Qualitative Crosslinking Test

During the solutions' incubation it was observed that solution Bunderwent crosslinking after 1 min, while solution A did not undergocrosslinking even after 2 hr. Therefore succinylation of the gelatinclearly blocked cross-linking.

Example 24 Succinylated Gelatin can be Mixed with Non-Modified Gelatinin a Manner that Improves the Mechanical Properties of a CrosslinkedGelatin Composition Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), 10% microbial Transglutaminase—ACTIVA-TG 10%(10% enzyme, 90% maltodextrin) (Ajinomoto, Japan), Succinylated gelatinwhich was prepared as previously described.

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, were prepared.

21% (w/v) Gelatin solution with Succinylated gelatin solution and 3.8MUrea, 0.15M Ca, 0.1M Sodium Acetate solution (Solution A), 21% (w/v)Gelatin solution with distilled water and 3.8M Urea, 0.15M Ca, 0.1MSodium Acetate solution (Solution B), 0.75% (w/w) mTG in 0.2M Na-Citrate(solution 1) were prepared.

The mTG crosslinking time of solutions A and B were tested byviscometer. For each viscometry test, 20 mL of gelatin solution wasmixed with 10 mL of mTG solution in a 50 mL beaker. The viscosity of themixed gelatin-mTG solution was then tracked as it underwent gelation.Different test groups were compared by recording the time required foreach test group to achieve 90% of the maximum viscosity able to berecorded by the viscometer at the specific speed and with the specificspindle used for that test.

In this experiment, a DV II+ PRO Digital Viscometer (BrookfieldEngineering, Middleboro, Mass.) was used with a T-E 95 “t-bar” spindle.A helipath viscometer stand was used to maintain vertical movement ofthe spindle over the course of the viscometer test. The helipath movedalong a 1 cm path. The viscometer readings were outputted by theviscometer and read using HyperTerminal software at a rate of 1 readingper second. The rotational speed of the spindle for the viscometry testwas 0.5 rpm. The maximum recordable viscosity at this speed with the T-E95 spindle was 10×10⁶ cP, meaning that the 30% point was equivalent to3×10⁶ cP and the 90% point was equivalent to 9×10⁶ cP.

The beaker was submerged in a 37° C. water bath for the entire extent ofthe viscometer test. Average temperature within the beaker also recordedthroughout the test to ensure consistency between test groups.

Upon the completion of the viscometry test, the resulting crosslinkedgelatin plug was removed from the beaker and submerged in a saline bath.After 24 hours, both the Solution A and Solution B gelatin plugs wereremoved from the saline bath and manually palpated to assess comparativeflexibility.

Results

Table 29 describes results of crosslinking time, demonstrating that amixture of succinylated and non-succinylated has a longer timerequirement for crosslinking.

TABLE 29 Average crosslinking time, Composition of crosslinked gel minSolution A 2:17 Solution B 1:30

All three (3) gelatin plugs formed from solution A were noticeably moreflexible than all three (3) gelatin plugs formed from solution B,indicating that the inclusion of succinylated gelatin in a gelatinsolution reduces the brittleness of that solution after crosslinking bymTG.

Example 25 Use of Carbamylation to Block the Amine Group Substrates inGelatin

This example describes the use of carbamylation to improve theflexibility and elasticity of a crosslinked gelatin composition.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Sodium Cyanate 96% (Sigma-Aldrich, St.Louis), 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme, 90%maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1 M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, 0.3M Sodium Cyanate wereprepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(solution A), and 0.75% (w/w) mTG in 0.2M Na-Citrate (solution 1) wereprepared.

Viscometer Tests

Viscometer tests were carried out as described in Example 19.

Prior to mixing gelatin and enzyme, sodium Cyanate was added either tosolution A or 1 as follows:

Addition of 100-200 μl 0.3M Sodium Cyanate to 10 ml of solution 1. Themixture was inverted several times and then added to 20 ml solution A,and the viscometer test was performed.

Addition of 50-150 μl 0.3M Sodium Cyanate to 20 ml solution A. Themixture was inverted several times and then added to 10 ml solution 1,and the viscometer test was performed.

Results

Table 30 describes results of the viscometer tests. In addition toincreased crosslinking time, resulting from the partially blocked aminoside groups in the Lysine chains, qualitative observations indicate thatcrosslinked gels treated with Cyanate groups lead to a more flexible,elastic and adhesive crosslinked gel.

TABLE 30 Viscometer test results Cyanate final Description added Volumeof 0.3M crosslinking of crosslinked to Cyanate used, μl time, min gelafter 1 hour none 0 2:30-2:45 Very rigid and stiff, poor flexibility,poor adhesion solution 1 100 4:30 Fairly adhesive, fairly flexible,rigid solution 1 200 7:30 Good adhesion and flexibility. Amorphousshape. solution A 50 2:34 Good adhesion and flexibility solution A 752:58 Good adhesion and flexibility solution A 100 2:41 Very goodadhesion and flexibility. solution A 150 3:18 Very good adhesion andflexibility.

Example 26 Effect of a Diamine Compound, Putrescine, on the Kinetics ofa Gelatin Crosslinking Reaction

This Example demonstrates that petruscine slowed down the crosslinkingreaction in a dose dependent manner, suggesting that it serves as asubstrate for transglutaminase and is crosslinked with the gelatin.Furthermore, the resultant crosslinked gelatin was more elastic.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Putrescine dihydrochloride (Sigma-Aldrich,St. Louis), 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme,90% maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, were prepared.

25% (w/w) Gelatin in 3.8M Urea, 0.15M CaCl2, 0.1M Sodium Acetate(control solution), 25% (w/w) Gelatin with 1-1 molar ratio of Putrescinein 3.8M Urea, 0.15M CaCl2, 0.1M Sodium Acetate (Solution A), 25% (w/w)Gelatin with 1½ molar ratio of Putrescine in 3.8M Urea, 0.15M CaCl2,0.1M Sodium Acetate (Solution B), 25% (w/w) Gelatin with 1¼ molar ratioof Putrescine in 3.8M Urea, 0.15M CaCl2, 0.1M Sodium Acetate (SolutionC), 1.25% (w/w) mTG in 0.2M Na-Citrate (solution 1) 0.75% (w/w) mTG in0.2M Na-Citrate (solution 2), 0.5% (w/w) mTG in 0.2M Na-Citrate(solution 3) and 0.25% (w/w) mTG in 0.2M Na-Citrate (solution 4) wereprepared.

Solutions were examined using Qualitative and Elasticity tests.

Qualitative Crosslinking Test

Solutions A, B and C with solutions 1 and 2 were mixed in 2:1 ratio,then moved to 37 C incubator and crosslinking time was defined when anapparent gelation was noticed.

Elasticity Test

The control solution and Solution B were examined for changes inelasticity over time, using mTG3 and mTG4, respectively.

For each elasticity test, 6 ml of gelatin solution were mixed with 3 mlof mTG solution. The resulting mixture was applied to dog-bone mold, 2ml in each pattern. Molds are incubated in 370 C for 10 min. After theincubation, molds are covered in saline and extracted from the mold.

The specimen is either incubated in saline in RT, or examinedimmediately. Pattern thickness is measured, and the specimen is placedbetween the instrument's clamps.

Results

Qualitative Crosslinking Test

Table 31 displays the results for the qualitative crosslinking test,showing that increasing concentrations of putrescine results inincreased cross-linking times.

TABLE 31 crosslinking time of the samples Gelation time Gelatin SolutionmTG solution [min] A 2 4:00 B 2 3:00 C 2 2:00 A 1 2:40 B 1 2:00 control1 1:40

Elasticity Test

Table 32 displays the average results for the elasticity test; theresults are for 2 hr incubation in saline in RT.

TABLE 32 elasticity test results Gelatin Modulus Tensile Stress TensileStrain at Solution mTG solution (kPa) at Break (kPa) Break (%) Control 379.01 49.78 62.1 B 4 49.18 42.61 88.05

Example 27 Use of an Amine Donor, Polyethylenimine (PEI), to Increasethe Elasticity of a mTG-Crosslinked Gelatin Composition

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), Urea 99.5% (Sigma-Aldrich,St. Louis), Calcium Chloride (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), Polyethylenimine (PEI) Mw 750,000 (Sigma,St. Louis, Mo.), 10% microbial Transglutaminase—ACTIVA-TG 10% (10%enzyme, 90% maltodextrin) (Ajinomoto, Japan).

Methods

Stock solutions of 0.1M Sodium Acetate buffer pH 6.0, 0.2M SodiumCitrate buffer pH 6.0, 4.5M Urea, 2M CaCL2, were prepared.

25% (w/w) Gelatin solution in 3.8M Urea, 0.15M Ca, 0.1M Sodium Acetate(solution A), 0.75% (w/w) mTG in 0.2M Na-Citrate (solution 1), 0.75%(w/w) mTG with 10% v/v PEI in 0.2M Na-Citrate (solution 2), 0.25% (w/w)mTG in 0.2M Na-Citrate (solution 3) and 0.25% (w/w) mTG with 10% v/v PEIin 0.2M Na-Citrate (solution 4) were prepared.

Viscometer Tests

Viscometer tests were carried out as described in Example 19.

Mechanical Testing with Instron

Mechanical properties, for material characterization, were tested withthe Instron device.

For each elasticity test, 6 ml of gelatin solution were mixed with 3 mlof mTG solution. The resulting mixture was applied to dog-bone mold, 2ml in each pattern. The molds were incubated in 37 C for 10 min. Afterthe incubation, the molds were covered in saline and extracted from themold.

The specimen was either incubated in saline in room temperature (RT), orexamined immediately. Pattern thickness was measured, and the specimenwas placed between the instrument's clamps.

Modulus, Tensile Stress at Break and Tensile Strain at Break (degree ofelongation) wee calculated for each specimen.

Results

Table 32 summarizes results of solution A which was crosslinked by mTGsolutions with and without PEI. Both viscometer and Instron tests werecarried out for achieving crosslinking time and mechanical properties ofthe crosslinked gels. Instron test was carried out after two hours ofincubation.

Results indicate that crosslinking time of solution A with mTGcontaining PEI (solution 2) slightly increases by 7%-14% compared to useof mTG solution with no PEI (solution 1).

Mechanical properties of crosslinked gels containing PEI were muchimproved as can be seen from comparison between Al with solution 3 and Awith solution 4; Modolus of the PEI based crosslinked gel was by 10%lower, and achieved elongation greater by more than 60% compared tocrosslinked gel with no PEI, as shown in Table 33.

TABLE 33 Viscometer and Instron tests for gelatin solution reacted withmTG with and without addition of 10% v/v PEI. Instron tests wereperformed after 2 hours incubation in RT Average Average CrosslinkingAverage Tensile Average Solution A time, min Modulus, Stress at Break,Tensile Strain reacted with (30%, 90%) kPa kPa at Break, % solution 11:54, 2:35 — — — solution 2 2:03, 2:58 — — — solution 3 — 79 50 62solution 4 — 72 74 103

Example 28 Dispersal of Raw mTG Powder into Foamed Gelatin

This example describes the formation of an appropriate gelatin foam andthe dispersal of dry mTG into the foam such that no reaction isimmediately detected. Only once the dry composition is reconstituted iscrosslinking activity shown.

Materials

The following materials were used in the experiment: Gelita 275 bloom,type A porcine gelatin (Gelita, Sioux City), WFI (Water For Injection;Cure, Israel), 10% microbial Transglutaminase—ACTIVA-TG 10% (10% enzyme,90% maltodextrin) (Ajinomoto, Japan).

Methods

Gelatin solution for foaming was chosen as 5% w/w in WFI water (thisconcentration was proved to achieve good gelatin structure while thesolvent was chosen as WFI so the Eutectic point will be relative high).The Gelatin solution was maintained at 36-37 Celsius prior to foamingwhile environment temperature was 21 C-22 C. Gelatin solution at 36 C-37C was loaded and foamed by a mechanical device. After foaming procedurewas complete, 15 g of foam were loaded on each Aluminum tray and then0.25 g of mTG raw powder (with 90% Maltodextrin) was dispersed with asieve onto the foam layer. Then additional 15 g of foam were loaded andcovered the mTG layer on each tray to achieve total 30 g of foam withless than 1% w/w mTG integrated in every tray. Because it has shownbefore that raw mTG powder does not react immediately with its substratedue to its low solubility, no cross-linking reaction was observed whileloading the trays (no stiffening was detected). All trays weretransferred and loaded in to the Freeze Dryer shelves. The testingprogram started only after all trays were inside the FD, All trays werestored in the same final temperature environment (−40 Celsius). Overalltime of the freeze drying program was 36-40 hours.

Reconstitution Test:

The lyophilized pads were extracted from the treys and submerged inwater to observe for reconstitution process.

These tests revealed that large areas within the foam matrix did notdissolve. These findings, along with the observation of a typical greycolor of the mTG powder in those areas, indicate that part of the foammatrix underwent cross linking reaction and therefore did not dissolveeven after 20 minutes.

Example 29 mTG Crosslinking of Recombinant Gelatin Materials

100 kDa recombinant gelatin (rGelatin) in lyophilized chunks,approximately 2 cm in thickness. ACTIVA TG microbial transglutaminasepowder (10% protein, 90% maltodextrin; Ajinomoto, Japan). PhosphateBuffered Saline (PBS), pH 7.4.

Methods

PBS is brought to room temperature (23-25° C.). A 25% w/w solution ofrGelatin in PBS is prepared by manually stirring rGelatin into ahomogenous solution. Once the solution is thoroughly mixed, it isbriefly centrifuged to remove air.

A 7.5% w/w solution of ACTIVA TG in PBS is prepared by manually stirringmTG into a homogenous Solution (mTG solution).

2 mL of rGelatin solution and 1 mL of mTG solution are dispensed into aplastic weighing dish using pipettes. Immediately following dispensing,solutions are thoroughly mixed using a pipette tip. The mixed solutionis palpated using a pipette tip every 30 seconds to qualitatively assesstime of gelation. Once gelation is observed, the sample is manuallyremoved from plate and the elasticity of sample is demonstrated bymanually stretching.

The above sample preparation procedure is repeated and the sample isplaced in a beaker in a 50° C. bath for 10 minutes. The sample is thensubjectively assessed to determine whether it maintains its gel phase orreturns to a liquid phase (i.e. to assess thermoreversibility).

Expected Results

It is expected after this process that gelation is observed such thatrGelatin is expected to function as a substrate for microbialtransglutaminase crosslinking.

Example 30 mTG Crosslinking of Type B Gelatin

Previous efforts have been made to use mTG to crosslink Type B gelatin.However, while physical gelation of type B gelatin has been recorded;efforts at mTG-crosslinking of type B gelatin has not been successful(Crescenzi et al. Biomacromolecules. 2002, 3: p. 1384-1391).Surprisingly, it was found that in an embodiment of, the hereininvention, where the gelatin was dissolved in acetate buffer and the mTGin citrate buffer, mTG crosslinking of type B gelatin resulted in theformation of a vigorous gel.

Materials

225 Bloom, Type B bovine gelatin (Sigma, St. Louis, Mo.), Sodium Acetatetrihydrate (Sigma-Aldrich, St. Louis), Acetic Acid 100% (Ridel-De Haen),Sodium Citrate (Sigma-Aldrich, St. Louis), Citric Acid Monohydrate(Sigma-Aldrich, St. Louis), ACTIVA TG microbial transglutaminase powder(10% protein, 90% maltodextrin; Ajinomoto, Japan).

Methods

The following solutions were prepared: 25% (w/w) gelatin solution in0.1M Sodium Acetate pH 6.0 at 50° C., 7.5% (w/w) ACTIVA solution in 0.2MNa-Citrate (mTG solution).

A portion of gelatin solution was incubated at room temperature (22-23°C.) and temperature was tracked as it dropped from 50° C. Transitionpoint from liquid solution to physical gel was determined by stirringthe gelatin solution periodically as temperature dropped. Physicalgelation point was determined as temperature at which stirrer could nolonger be used to displace gelatin solution.

Separately, 2 mL aliquots of gelatin solution at 50° C. were mixed witheither 1 mL or 2 mL of mTG solution in plastic tubes. These tubes wereplaced in an incubator at 37° C. The tubes were removed every 30 secondsand inverted to determine if enzymatically crosslinked gel had beenformed. Gelation time was defined as the time when the gelatin-mTGmixture no longer flowed upon tube inversion.

Results

The physical sol-gel transition point of the gelatin solution occurredat 30° C. Gelation was observed in both gelatin-mTG compositions within2 minutes.

These results indicate that type B gelatin can form both athermoreversible physical gel below about 30° C. and an mTG-crosslinkedchemical gel at higher temperatures.

Example 31 Burst Pressure Adhesive Tests Materials

The materials used for this experiment were type A, 300 bloom, 70 meshporcine pharmaceutical gelatin (Medex, England batch #80067), 98% urea,dried powder (Alfa Aesar, Lancester: Lot #10110586), 97% CaCl₂, driedpowder (Alfa Aesar, Lancester: Lot #10110561), 0.1M Sodium Acetatebuffer (pH 6.1), 0.5M Sodium Citrate buffer, D-Sorbitol, 97% (Sigma, St.Louis, Mo.: batch #1344776), 10% microbial Transglutaminase—ACTIVA-TG10% (10% enzyme, 90% maltodextrin) [Ajinomoto, Japan].

Methods

Gelatin Solutions Preparation:

-   -   1. Control—25% (w/w) gelatin solution in 0.1 M sodium acetate        buffer.    -   2. 25% (w/w) gelatin, 4.5M urea in 0.1M sodium acetate buffer.    -   3. 25% (w/w) gelatin, 2M urea 1M Ca in 0.1M sodium acetate        buffer.

In order to completely dissolve the gelatin powders, the solutions wereheated to 40° C. and vigorously stirred. The solutions were then kept ina 24° C. incubator overnight prior to experiment.

Microbial Transglutaminase Solutions Preparation:

Sodium Acetate Solutions:

-   -   a. 5% (w/w) ACTIVA-TG 10% in 0.1M sodium acetate with sorbitol        added to a 3:1 w/w ratio, sorbitol:gelatin.    -   b. 5% (w/w) ACTIVA-TG 10% in 0.25M sodium acetate with sorbitol        added to a 3:1 w/w ratio, sorbitol:gelatin.    -   c. 7.5% (w/w) ACTIVA-TG 10% in 0.1M sodium acetate with sorbitol        added to a 3:1 w/w ratio, sorbitol:gelatin.    -   d. 7% (w/w) ACTIVA-TG 10% in 0.1M sodium acetate.    -   e. 7.5% (w/w) ACTIVA-TG 10% in 0.1M sodium acetate with sorbitol        added to a 3:1 w/w ratio, sorbitol:gelatin.

Sodium Citrate Solutions:

-   -   f. 2.5% (w/w) ACTIVA-TG 10% in 0.1M sodium citrate.    -   g. 3% (w/w) ACTIVA-TG 10% in 0.1M sodium citrate.    -   h. 10% (w/w) ACTIVA-TG 10% in 0.1M sodium citrate.    -   i. 5% (w/w) ACTIVA-TG 10% in 0.5M sodium citrate with sorbitol        added to a 3:1 w/w ratio, sorbitol:gelatin.    -   j. 10% (w/w) ACTIVA-TG 10% in 0.5M sodium citrate with sorbitol        added to a 3:1 w/w ratio, sorbitol:gelatin.    -   k. 5% (w/w) ACTIVA-TG 10% in 0.5M sodium citrate with sorbitol        added to a 0.6:1 w/w ratio, sorbitol:gelatin.    -   Enzyme solutions were filtered using whatman filter paper no. 1        prior to use.

Burst Pressure Tests:

Burst pressure tests were run according to a modified form of ASTMprotocol designation F 2392-04.

Burst pressure adhesive tests were run to compare the adhesiveness ofdifferent adhesive compositions. For each burst pressure test, collagensausage casing (Nitta Casings, New Jersey) was used as a substrate. A2-mm diameter hole was punched in each casing specimen and each specimenwas clamped into the specimen holding manifold. Then, the tissuemanifold was filled with heated (37° C.) physiological fluid (salinesolution). Once the manifold was filled, an experimental cross-linkingcomposition was prepared and applied on top of the specimen hole tocompletely cover the hole with the composition.

Experimental compositions were prepared by mixing 4 mL of cross-linkableprotein solution with 2 mL of non-toxic cross-linker in a 25 mL beaker.Then, 4 mL of the mixed solution was pulled into a 5 mL syringe. Aftergelatin and mTG solutions were mixed in a small beaker, 4 ml of theformed-gel was transferred into a syringe. Then, 3 mL of the compositionwas applied to cover the specimen hole. After 4 minutes (from the momentof mixing), the burst pressure system was activated and pressure wasbuilt up to the indicated level below the hole until the adhesivecomposition failed and the fluid burst through the specimen hole.

In order to achieve reproducibility, each formulation was tested atleast 4 times, unless mentioned otherwise.

Schematics of the burst pressure system and the associated tissuemanifold are shown in FIG. 9.

Results

The following tables summarize results of the burst pressure tests ofthe different gelatin-mTG combinations. The initial atmospheric pressurewas measured to be 755 mmHg. Thus, in all cases, the net pressureexperienced by the gelatin-mTG composition adhered to the substrate wasequal to the recorded pressure value (noted in the results tables) minus755 mmHg. For example, a recorded value of 855 mmHg indicates that theadhesive strength of the composition being tested in the burst pressuresystem was 100 mmHg.

Control—25% (w/w) gelatin in 0.1M Na—Ac solution

TABLE 34 Summary of tests using 25% (w/w) gelatin in 0.1M Na—Ac solutionwith mTG # mTG % mTG in experiment solution solution Summary of tests 10.1M 7 Test 1 - pressure applied was slowly Na—Ac raised to 870 mmHg.After 4 minutes in that pressure was raised again but test was failed in884 mmHg. Cohesive failure from center of formed gel was noticed.Overall time - 8:30 minutes.25% (w/w) gelatin, 4.5M urea in 0.1M Na—Ac solution

TABLE 35 Summary of tests using 25% (w/w) gelatin, 4.5M urea in 0.1MNa—Ac solution with different mTG based solutions # mTG % mTG inexperiment solution solution Summary of tests 2 0.1M 7 Test 1 - Formedgel maintained stability in 820-837 mmHg Na—Ac for 1 minute and failedafterwards (cohesive failure). Overall time - 6 minutes. Test 2 -pressure of 820-828 was maintained for 2 minutes. Applied pressure wasraised afterwards and the cross-linked gel failed (cohesive) in 851mmHg. Overall time - 8 minutes. Test 3 - Similar to test 2. Gelmaintained integrity in range of 820-837 mmHg for 2 minutes. Failedafterwards (cohesive failure) when pressure was raised to 853. Test 4 -Formed gel failed in 834 mmHg. Overall time - 4:52. Test 5 - Adhesiveand cohesive failure occurred in 808 mmHg (bubble formation of gel wasnoticed). Test 6 - Unlike other tests, pressure was applied only after42 minutes (instead of 4) and pressure was slowly raised. Formed gelfailed in 906 mmHg (cohesive failure) after overall time of 46 minutes.2 0.5M 2.5 Test 1 - an almost immediate failure occurred, in Na- 770mmHg. Failure was both cohesive and Citrate adhesive. Test 2 - Similarto results of test 1. Bubble formation of the formed gel was noticed.Test 3 - pressure was applied only after 5 minutes, however as inprevious tests an almost immediate failure, due to bubble formation,occurred. Cohesive and adhesive failure took place in 790 mmHg. Overalltime - minutes. 3 0.1M 7.5% Test 1 - cohesive failure was occurredduring Na—Ac + pressure raise in 807 mmHg. Overall time - 5 sorbitolminutes. 3:1 Test 2 - as in test 1. Combined failure (cohesive andadhesive) occurred in 817 mmHg. Bubble formation was noticed. Test 3 -much alike test 2. Cohesive failure occurred in 813 mmHg. Overall time -4:46 minutes. Test 4 - as oppose to previous tests, this time thegelatin and mTG solutions were mixed on the substrate itself. Whenpressure was applied bubble formation in the formed gel occurred and itfailed in 804 mmHg. Overall time - 5:20 minutes. Test 5 - formed gelmaintained integrity in 820-833 mmHg for 2 minutes. When pressure wasraised again, failure occurred in 843 mmHg. Overall time - 8:15. Test6 - Combined cohesive and adhesive failure occurred in 825 mmHg. Overalltime - 5:20 minutes. Test 7 - bubble formation caused to an combinedcohesive and adhesive failure in 791 mmHg. Overall time - 5:08 minutes.4 0.5M 5 Test 1 - since cross-linked was formed almost Na- immediately;application on the substrate was not Citrate + uniformed and caused manyair bubbles. As a sorbitol result, failure occurred in the pressureraised 3:1 sequence and it was also noted that the adhesion was poor.Test 2 - both solutions were mixed inside the syringe and was applieddirectly on the substrate but result resembled test 1 as failureoccurred in the pressure raised stage. Once again, bubble formation wasnoticed. 5 0.1M 7.5 Test 1 - cohesive failure through center of gel, atNa—Ac 808 mmHg. Overall time - 4:50. Good adhesion to the substrate wasnoticed. Test 2 - test was held while slightly tilting the system, inorder to prevent air bubbles gather under the substrate. Cohesivefailure occurred at 847 mmHg. Overall time - 5:30. Good adhesion wasreported. Test 3 - cohesive failure occurred at 811 mmHg. Overall time -4:50.25% (w/w) gelatin, 2M urea 1M Ca in 0.1M Na—Ac solution

TABLE 36 Summary of tests using 25% (w/w) gelatin, 2M urea 1M Ca in 0.1MNa—Ac solution with different mTG based solutions # mTG % mTG inexperiment solution solution Summary of tests 6 0.1M 7.5 Test 1 - animmediate failure in 760 mmHg. Na—Ac Test 2 - pressure was raised to 820mmHg and formed gel maintained integrity for 0:30 minutes. Cohesivefailure was noticed although gel possessed poor adhesion as well. Test3 - much like test 1. Poor adhesion was noticed as well. Test 4 -pressure was raised to 820 mmHg and formed gel maintained integrity for0:40 minutes. Cohesive failure was noticed although gel possessed pooradhesion as well. 7 0.5M 10 Test 1 - pressure was raised to 820 mmHg andNa- cross-linked gel maintained formation for 2 Citrate minutes. Whenpressure was raised again, cohesive failure occurred in 840 mmHg. Pooradhesion of the gel to the substrate was noticed. Overall time - 10minutes. Test 2 - cohesive failure occurs while pressure was raised, in800 mmHg. Overall time - 4:30 minutes. Test 3 - cohesive failure occurswhile pressure was raised, in 808 mmHg. Overall time - 5:30 minutes.Test 4 - adhesive failure occurs while pressure was raised, in 808 mmHg.Overall time - 5 minutes. 8 0.1M 7.5 Test 1 - an almost immediatefailure due to bubble Na—Ac formation. Failure was reported as cohesiveand adhesive, though adhesion was noticed as fairly good. Overall time -4:20. Test 2 - same as in test 1.

Similar burst pressure results were obtained with other embodiments ofthe herein inventions.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1-12. (canceled)
 13. A composition comprising a cross-linkable proteinor polypeptide, with the proviso that said protein or polypeptide is notfibrin, an enzyme which induces cross-linking of said cross-linkableprotein, a metal ion and a denaturing agent, wherein said cross-linkableprotein or polypeptide comprises gelatin, wherein said enzyme comprisestransglutaminase, wherein said metal ion comprises calcium as anypharmaceutically compatible salt and wherein said denaturing agentcomprises urea.
 14. (canceled)
 15. The composition of claim 13, whereinsaid gelatin is at least 250 bloom.
 16. (canceled)
 17. The compositionof claim 15, wherein said calcium salt comprises one or more of calciumchloride or calcium hydroxide.
 18. The composition of claim 17, whereinsaid calcium is present in an amount of up to 1M.
 19. (canceled) 20.(canceled)
 21. The composition of claim 18, wherein said urea is presentin an amount of up to 4M.
 22. (canceled)
 23. The composition of claim21, further comprising a calcium sequestering agent when said metal ioncomprises calcium.
 24. (canceled)
 25. The composition of claim 23,wherein said calcium sequestering agent comprises one or more of EDTA,citrate or calgon.
 26. The composition of claim 25, wherein said citrateor said EDTA is present in an amount of from about 0.01 M to about 2M.27. The composition of claim 26, wherein said citrate or said EDTA ispresent in an amount from about 0.05M to about 0.2M. 28-32. (canceled)33. The composition of claim 26, further comprising a PEG (polyethyleneglycol) derivative capable of covalently binding to said gelatin. 34.The composition of claim 33, wherein said PEG derivative comprises anyaminated PEG derivative.
 35. (canceled)
 36. The composition of claim 26,further comprising a PVA (polyvinyl alcohol) derivative capable ofcovalently binding to said gelatin.
 37. The composition of claim 36,wherein said PVA derivative comprises any aminated PVA derivative.38-58. (canceled)
 59. The composition of claim 26, further comprisingacetate.
 60. The composition of claim 26, further comprising aplasticizer.
 61. The composition of claim 60, wherein said plasticizeris selected from the group consisting of Gum Arabic, Guar Gum, PVA, PEG6000, Polyvinylpyrrolidone (PVP), citric acid alkyl esters, glycerolesters, phthalic acid alkyl esters, sebacic acid alkyl esters, sucroseesters, sorbitan esters, sorbitol, acetylated monoglycerides, glycerols,fatty acid esters, glycols, propylene glycol, lauric acid, sucrose,glyceryl triacetate, poloxamers, diethyl phthalate, mono- anddi-glycerides of edible fats or oils, dibutyl phthalate, dibutylsebacate, polysorbate, polyethylene glycols 200 to 12,000, Carbowaxpolyethylene glycols, and a surfactant at a concentration above the CMC(critical micelle concentration) of said surfactant; or a combinationthereof.
 62. The composition of claim 61, wherein said surfactantcomprises a polyoxyethylene-sorbitan-fatty acid ester,polyoxyethyleneglycol dodecyl ether, polyoxyethylene-polyoxypropyleneblock copolymer, sodium lauryl sulfate, sodium dodecyl sulfate, sodiumlaureth sulfate, sodium lauryl ether sulfate, poloxamers, poloxamines,alkyl polyglucosides, fatty alcohols, fatty acid salts, cocamidemonoethanolamine, and cocamide diethanolamine.
 63. The composition ofclaim 62, wherein a concentration of said surfactant is in the range offrom about 0.1% to about 5% w/w of dry weight of said cross-linkableprotein.
 64. The composition of claim 63, wherein saidpolyoxyethylene-sorbitan-fatty acid ester comprises one or more ofpolysorbates 20, 21, 0, 60, 61, 65, 80 or
 85. 65. The composition ofclaim 26, further comprising a viscosity increasing agent selected fromthe group consisting of Alginate Ester, Gum Arabic, high viscosityCarboxymethyl cellulose (CMC), Xanthan Gum, Guar Gum, and PVP.
 66. Thecomposition of claim 26, further comprising one or more of Cystamine,Cysteine, cyanate or Melanin.
 67. The composition of claim 26, furthercomprising an ammonia scavenging, sequestering or binding agent, astimulator of ammonia metabolism, or an inhibitor of cellular ammoniauptake. 68-70. (canceled)
 71. The composition of claim 67, wherein saidstimulator of ammonia metabolism or said inhibitor of cellular ammoniauptake comprises L-glutamine, L-glutamate, or a combination thereof. 72.(canceled)
 73. (canceled)
 74. The composition of claim 26, having a pHin a range of from about 6 to about
 7. 75. The composition of claim 74,wherein said pH is about
 6. 76. (canceled)
 77. The composition of claim13, wherein said transglutaminase is calcium independent.
 78. Thecomposition of claim 77, wherein said transglutaminase is microbialtransglutaminase.
 79. The composition of claim 78, wherein a proteinconcentration of said transglutaminase is present in an amount fromabout 0.0001% to about 2% w/w of the composition.
 80. The composition ofclaim 79, wherein said transglutaminase is present in an amount of fromabout 0.01% to about 1.35% w/w of the composition.
 81. (canceled) 82.(canceled)
 83. The composition of claim 82, wherein said concentrationof transglutaminase is in the range of from about 1 to about 180 enzymeunits (U/mL) of total composition.
 84. (canceled)
 85. (canceled)
 86. Thecomposition of claim 78, wherein a ratio of enzyme solution to:gelatinsolution is about 1:1 to 1:5 v/v.
 87. The composition of claim 13,wherein said gelatin is produced from animal origin, recombinant originor a combination thereof.
 88. The composition of claim 87, wherein saidanimal origin is selected from the group consisting of fish and mammals.89. (canceled)
 90. The composition of claim 89, wherein said gelatin isof type A (Acid Treated) or of type B (Alkaline Treated).
 91. (canceled)92. The composition of claim 91, wherein said gelatin comprises highmolecular weight gelatin of at least about 250 bloom, or equivalentthereof. 93-108. (canceled)
 109. The composition of claim 13, wherein anamount of said urea is reduced by a percentage in a range of from 30% to50% in a presence of said calcium, in comparison to an amount of saidurea in an absence of said calcium.